Ligand binding by repeat proteins: natural and designed

PubMed Central

Grove, Tijana Z; Cortajarena, Aitziber L; Regan, Lynne

2012-01-01

Repeat proteins contain tandem arrays of small structural motifs. As a consequence of this architecture, they adopt non-globular, extended structures that present large, highly specific surfaces for ligand binding. Here we discuss recent advances toward understanding the functional role of this unique modular architecture. We showcase specific examples of natural repeat proteins interacting with diverse ligands and also present examples of designed repeat protein–ligand interactions. PMID:18602006

Characterization of two genes encoding leucine-rich repeat-containing proteins in grass carp Ctenopharyngodon idellus.

PubMed

Chang, M X; Nie, P; Xie, H X; Sun, B J; Gao, Q

2005-01-01

The cDNAs and genes of two different types of leucine-rich repeat-containing proteins from grass carp (Ctenopharyngodon idellus) were cloned. Homology search revealed that the two genes, designated as GC-GARP and GC-LRG, have 37% and 32% deduced amino-acid sequence similarities with human glycoprotein A repetitions predominant precursor (GARP) and leucine-rich alpha2-glycoprotein (LRG), respectively. The cDNAs of GC-GARP and GC-LRG encoded 664 and 339 amino acid residues, respectively. GC-GARP and GC-LRG contain many distinct structural and/or functional motifs of the leucine-rich repeat (LRR) subfamily, such as multiple conserved 11-residue segments with the consensus sequence LxxLxLxxN/CxL (x can be any amino acid). The genes GC-GARP and GC-LRG consist of two exons, with 4,782 bp and 2,119 bp in total length, respectively. The first exon of each gene contains a small 5'-untranslated region and partial open reading frame. The putative promoter region of GC-GARP was found to contain transcription factor binding sites for GATA-1, IRF4, Oct-1, IRF-7, IRF-1, AP1, GATA-box and NFAT, and the promoter region of GC-LRG for MYC-MAX, MEIS1, ISRE, IK3, HOXA9 and C/EBP alpha. Phylogenetic analysis showed that GC-GARP and mammalian GARPs were clustered into one branch, while GC-LRG and mammalian LRGs were in another branch. The GC-GARP gene was only detected in head kidney, and GC-LRG in the liver, spleen and heart in the copepod (Sinergasilus major)-infected grass carp, indicating the induction of gene expression by the parasite infection. The results obtained in the present study provide insight into the structure of fish LRR genes, and further study should be carried out to understand the importance of LRR proteins in host-pathogen interactions.

The evolution of filamin-a protein domain repeat perspective.

PubMed

Light, Sara; Sagit, Rauan; Ithychanda, Sujay S; Qin, Jun; Elofsson, Arne

2012-09-01

Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. Copyright © 2012 Elsevier Inc. All rights reserved.

The evolution of filamin – A protein domain repeat perspective

PubMed Central

Light, Sara; Sagit, Rauan; Ithychanda, Sujay S.; Qin, Jun; Elofsson, Arne

2013-01-01

Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. PMID:22414427

Armadillo Repeat Containing 8α Binds to HRS and Promotes HRS Interaction with Ubiquitinated Proteins

PubMed Central

Tomaru, Koji; Ueda, Atsuhisa; Suzuki, Takeyuki; Kobayashi, Nobuaki; Yang, Jun; Yamamoto, Masaki; Takeno, Mitsuhiro; Kaneko, Takeshi; Ishigatsubo, Yoshiaki

2010-01-01

Recently, we reported that a complex with an essential role in the degradation of Fructose-1,6-bisphosphatase in yeast is well conserved in mammalian cells; we named this mammalian complex C-terminal to the Lissencephaly type-1-like homology (CTLH) complex. Although the function of the CTLH complex remains unclear, here we used yeast two-hybrid screening to isolate Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) as a protein binding to a key component of CTLH complex, Armadillo repeat containing 8 (ARMc8) α. The association was confirmed by a yeast two-hybrid assay and a co-immunoprecipitation assay. The proline-rich domain of HRS was essential for the association. As demonstrated through immunofluorescence microscopy, ARMc8α co-localized with HRS. ARMc8α promoted the interaction of HRS with various ubiquitinated proteins through the ubiquitin-interacting motif. These findings suggest that HRS mediates protein endosomal trafficking partly through its interaction with ARMc8α. PMID:20224683

Structural and biophysical properties of h-FANCI ARM repeat protein.

PubMed

Siddiqui, Mohd Quadir; Choudhary, Rajan Kumar; Thapa, Pankaj; Kulkarni, Neha; Rajpurohit, Yogendra S; Misra, Hari S; Gadewal, Nikhil; Kumar, Satish; Hasan, Syed K; Varma, Ashok K

2017-11-01

Fanconi anemia complementation groups - I (FANCI) protein facilitates DNA ICL (Inter-Cross-link) repair and plays a crucial role in genomic integrity. FANCI is a 1328 amino acids protein which contains armadillo (ARM) repeats and EDGE motif at the C-terminus. ARM repeats are functionally diverse and evolutionarily conserved domain that plays a pivotal role in protein-protein and protein-DNA interactions. Considering the importance of ARM repeats, we have explored comprehensive in silico and in vitro approach to examine folding pattern. Size exclusion chromatography, dynamic light scattering (DLS) and glutaraldehyde crosslinking studies suggest that FANCI ARM repeat exist as monomer as well as in oligomeric forms. Circular dichroism (CD) and fluorescence spectroscopy results demonstrate that protein has predominantly α- helices and well-folded tertiary structure. DNA binding was analysed using electrophoretic mobility shift assay by autoradiography. Temperature-dependent CD, Fluorescence spectroscopy and DLS studies concluded that protein unfolds and start forming oligomer from 30°C. The existence of stable portion within FANCI ARM repeat was examined using limited proteolysis and mass spectrometry. The normal mode analysis, molecular dynamics and principal component analysis demonstrated that helix-turn-helix (HTH) motif present in ARM repeat is highly dynamic and has anti-correlated motion. Furthermore, FANCI ARM repeat has HTH structural motif which binds to double-stranded DNA.

[Study on nano-CaCO3 applicated in Xin Yue Shu Capsules preliminarily].

PubMed

Jiang, Yan-Rong; Zhang, Zhen-Hai; Cui, Li; He, Jun-Jie; Hu, Shao-Ying; Jia, Xiao-Bin

2012-11-01

To investigate the characteristics of nano-CaCO3 applicated in Xin Yue Shu Capsules. Studied the effect of different dosages of aerosil or nano-CaCO3 on fluidity, bulk density, moisture absorption of Xin Yue Shu capsules spray drying powder. In vitro dissolution and ferulic acid stability of Xin Yue Shu capsules was observed. It significantly improved powder fluidity and bulk density of Xin Yue Shu spray drying powder when aerosil or nano-CaCO3 was added. But there was no significant effect on powder moisture absorption, ferulic acid in vitro dissolution and ferulic acid stability. The effect of Nano-CaCO3 on improving powder fluidity and bulk density applicated in the spray drying powder of traditional Chinese medicine deserves studying further.

Topological characteristics of helical repeat proteins.

PubMed

Groves, M R; Barford, D

1999-06-01

The recent elucidation of protein structures based upon repeating amino acid motifs, including the armadillo motif, the HEAT motif and tetratricopeptide repeats, reveals that they belong to the class of helical repeat proteins. These proteins share the common property of being assembled from tandem repeats of an alpha-helical structural unit, creating extended superhelical structures that are ideally suited to create a protein recognition interface.

Exploring the repeat protein universe through computational protein design

DOE PAGES

Brunette, TJ; Parmeggiani, Fabio; Huang, Po-Ssu; ...

2015-12-16

A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. In this paper, we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix–loop–helix–loop structural motif. Eighty-three designs with sequences unrelatedmore » to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Finally, our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.« less

Sequence repeats and protein structure

NASA Astrophysics Data System (ADS)

Hoang, Trinh X.; Trovato, Antonio; Seno, Flavio; Banavar, Jayanth R.; Maritan, Amos

2012-11-01

Repeats are frequently found in known protein sequences. The level of sequence conservation in tandem repeats correlates with their propensities to be intrinsically disordered. We employ a coarse-grained model of a protein with a two-letter amino acid alphabet, hydrophobic (H) and polar (P), to examine the sequence-structure relationship in the realm of repeated sequences. A fraction of repeated sequences comprises a distinct class of bad folders, whose folding temperatures are much lower than those of random sequences. Imperfection in sequence repetition improves the folding properties of the bad folders while deteriorating those of the good folders. Our results may explain why nature has utilized repeated sequences for their versatility and especially to design functional proteins that are intrinsically unstructured at physiological temperatures.

A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family.

PubMed

Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L; Salt, Jennifer N; Goring, Daphne R

2004-01-01

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis.

De novo identification of highly diverged protein repeats by probabilistic consistency.

PubMed

Biegert, A; Söding, J

2008-03-15

An estimated 25% of all eukaryotic proteins contain repeats, which underlines the importance of duplication for evolving new protein functions. Internal repeats often correspond to structural or functional units in proteins. Methods capable of identifying diverged repeated segments or domains at the sequence level can therefore assist in predicting domain structures, inferring hypotheses about function and mechanism, and investigating the evolution of proteins from smaller fragments. We present HHrepID, a method for the de novo identification of repeats in protein sequences. It is able to detect the sequence signature of structural repeats in many proteins that have not yet been known to possess internal sequence symmetry, such as outer membrane beta-barrels. HHrepID uses HMM-HMM comparison to exploit evolutionary information in the form of multiple sequence alignments of homologs. In contrast to a previous method, the new method (1) generates a multiple alignment of repeats; (2) utilizes the transitive nature of homology through a novel merging procedure with fully probabilistic treatment of alignments; (3) improves alignment quality through an algorithm that maximizes the expected accuracy; (4) is able to identify different kinds of repeats within complex architectures by a probabilistic domain boundary detection method and (5) improves sensitivity through a new approach to assess statistical significance. Server: http://toolkit.tuebingen.mpg.de/hhrepid; Executables: ftp://ftp.tuebingen.mpg.de/pub/protevo/HHrepID

Distribution and Evolution of Yersinia Leucine-Rich Repeat Proteins

PubMed Central

Hu, Yueming; Huang, He; Hui, Xinjie; Cheng, Xi; White, Aaron P.

2016-01-01

Leucine-rich repeat (LRR) proteins are widely distributed in bacteria, playing important roles in various protein-protein interaction processes. In Yersinia, the well-characterized type III secreted effector YopM also belongs to the LRR protein family and is encoded by virulence plasmids. However, little has been known about other LRR members encoded by Yersinia genomes or their evolution. In this study, the Yersinia LRR proteins were comprehensively screened, categorized, and compared. The LRR proteins encoded by chromosomes (LRR1 proteins) appeared to be more similar to each other and different from those encoded by plasmids (LRR2 proteins) with regard to repeat-unit length, amino acid composition profile, and gene expression regulation circuits. LRR1 proteins were also different from LRR2 proteins in that the LRR1 proteins contained an E3 ligase domain (NEL domain) in the C-terminal region or an NEL domain-encoding nucleotide relic in flanking genomic sequences. The LRR1 protein-encoding genes (LRR1 genes) varied dramatically and were categorized into 4 subgroups (a to d), with the LRR1a to -c genes evolving from the same ancestor and LRR1d genes evolving from another ancestor. The consensus and ancestor repeat-unit sequences were inferred for different LRR1 protein subgroups by use of a maximum parsimony modeling strategy. Structural modeling disclosed very similar repeat-unit structures between LRR1 and LRR2 proteins despite the different unit lengths and amino acid compositions. Structural constraints may serve as the driving force to explain the observed mutations in the LRR regions. This study suggests that there may be functional variation and lays the foundation for future experiments investigating the functions of the chromosomally encoded LRR proteins of Yersinia. PMID:27217422

Mechanical unfolding of an ankyrin repeat protein.

PubMed

Serquera, David; Lee, Whasil; Settanni, Giovanni; Marszalek, Piotr E; Paci, Emanuele; Itzhaki, Laura S

2010-04-07

Ankryin repeat proteins comprise tandem arrays of a 33-residue, predominantly alpha-helical motif that stacks roughly linearly to produce elongated and superhelical structures. They function as scaffolds mediating a diverse range of protein-protein interactions, and some have been proposed to play a role in mechanical signal transduction processes in the cell. Here we use atomic force microscopy and molecular-dynamics simulations to investigate the natural 7-ankyrin repeat protein gankyrin. We find that gankyrin unfolds under force via multiple distinct pathways. The reactions do not proceed in a cooperative manner, nor do they always involve fully stepwise unfolding of one repeat at a time. The peeling away of half an ankyrin repeat, or one or more ankyrin repeats, occurs at low forces; however, intermediate species are formed that are resistant to high forces, and the simulations indicate that in some instances they are stabilized by nonnative interactions. The unfolding of individual ankyrin repeats generates a refolding force, a feature that may be more easily detected in these proteins than in globular proteins because the refolding of a repeat involves a short contraction distance and incurs a low entropic cost. We discuss the origins of the differences between the force- and chemical-induced unfolding pathways of ankyrin repeat proteins, as well as the differences between the mechanics of natural occurring ankyrin repeat proteins and those of designed consensus ankyin repeat and globular proteins. Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The Energy Landscapes of Repeat-Containing Proteins: Topology, Cooperativity, and the Folding Funnels of One-Dimensional Architectures

PubMed Central

Komives, Elizabeth A.; Wolynes, Peter G.

2008-01-01

Repeat-proteins are made up of near repetitions of 20– to 40–amino acid stretches. These polypeptides usually fold up into non-globular, elongated architectures that are stabilized by the interactions within each repeat and those between adjacent repeats, but that lack contacts between residues distant in sequence. The inherent symmetries both in primary sequence and three-dimensional structure are reflected in a folding landscape that may be analyzed as a quasi–one-dimensional problem. We present a general description of repeat-protein energy landscapes based on a formal Ising-like treatment of the elementary interaction energetics in and between foldons, whose collective ensemble are treated as spin variables. The overall folding properties of a complete “domain” (the stability and cooperativity of the repeating array) can be derived from this microscopic description. The one-dimensional nature of the model implies there are simple relations for the experimental observables: folding free-energy (ΔGwater) and the cooperativity of denaturation (m-value), which do not ordinarily apply for globular proteins. We show how the parameters for the “coarse-grained” description in terms of foldon spin variables can be extracted from more detailed folding simulations on perfectly funneled landscapes. To illustrate the ideas, we present a case-study of a family of tetratricopeptide (TPR) repeat proteins and quantitatively relate the results to the experimentally observed folding transitions. Based on the dramatic effect that single point mutations exert on the experimentally observed folding behavior, we speculate that natural repeat proteins are “poised” at particular ratios of inter- and intra-element interaction energetics that allow them to readily undergo structural transitions in physiologically relevant conditions, which may be intrinsically related to their biological functions. PMID:18483553

Evolution of Protein Domain Repeats in Metazoa

PubMed Central

Schüler, Andreas; Bornberg-Bauer, Erich

2016-01-01

Repeats are ubiquitous elements of proteins and they play important roles for cellular function and during evolution. Repeats are, however, also notoriously difficult to capture computationally and large scale studies so far had difficulties in linking genetic causes, structural properties and evolutionary trajectories of protein repeats. Here we apply recently developed methods for repeat detection and analysis to a large dataset comprising over hundred metazoan genomes. We find that repeats in larger protein families experience generally very few insertions or deletions (indels) of repeat units but there is also a significant fraction of noteworthy volatile outliers with very high indel rates. Analysis of structural data indicates that repeats with an open structure and independently folding units are more volatile and more likely to be intrinsically disordered. Such disordered repeats are also significantly enriched in sites with a high functional potential such as linear motifs. Furthermore, the most volatile repeats have a high sequence similarity between their units. Since many volatile repeats also show signs of recombination, we conclude they are often shaped by concerted evolution. Intriguingly, many of these conserved yet volatile repeats are involved in host-pathogen interactions where they might foster fast but subtle adaptation in biological arms races. Key Words: protein evolution, domain rearrangements, protein repeats, concerted evolution. PMID:27671125

A Large Complement of the Predicted Arabidopsis ARM Repeat Proteins Are Members of the U-Box E3 Ubiquitin Ligase Family1[w

PubMed Central

Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L.; Salt, Jennifer N.; Goring, Daphne R.

2004-01-01

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis. PMID:14657406

Multiple functions of the leucine-rich repeat protein LrrA of Treponema denticola.

PubMed

Ikegami, Akihiko; Honma, Kiyonobu; Sharma, Ashu; Kuramitsu, Howard K

2004-08-01

The gene lrrA, encoding a leucine-rich repeat protein, LrrA, that contains eight consensus tandem repeats of 23 amino acid residues, has been identified in Treponema denticola ATCC 35405. A leucine-rich repeat is a generally useful protein-binding motif, and proteins containing this repeat are typically involved in protein-protein interactions. Southern blot analysis demonstrated that T. denticola ATCC 35405 expresses the lrrA gene, but the gene was not identified in T. denticola ATCC 33520. In order to analyze the functions of LrrA in T. denticola, an lrrA-inactivated mutant of strain ATCC 35405 and an lrrA gene expression transformant of strain ATCC 33520 were constructed. Characterization of the mutant and transformant demonstrated that LrrA is associated with the extracytoplasmic fraction of T. denticola and expresses multifunctional properties. It was demonstrated that the attachment of strain ATCC 35405 to HEp-2 cell cultures and coaggregation with Tannerella forsythensis were attenuated by the lrrA mutation. In addition, an in vitro binding assay demonstrated specific binding of LrrA to a portion of the Tannerella forsythensis leucine-rich repeat protein, BspA, which is mediated by the N-terminal region of LrrA. It was also observed that the lrrA mutation caused a reduction of swarming in T. denticola ATCC 35405 and consequently attenuated tissue penetration. These results suggest that the leucine-rich repeat protein LrrA plays a role in the attachment and penetration of human epithelial cells and coaggregation with Tannerella forsythensis. These properties may play important roles in the virulence of T. denticola.

New paradigm in ankyrin repeats: Beyond protein-protein interaction module.

PubMed

Islam, Zeyaul; Nagampalli, Raghavendra Sashi Krishna; Fatima, Munazza Tamkeen; Ashraf, Ghulam Md

2018-04-01

Classically, ankyrin repeat (ANK) proteins are built from tandems of two or more repeats and form curved solenoid structures that are associated with protein-protein interactions. These are short, widespread structural motif of around 33 amino acids repeats in tandem, having a canonical helix-loop-helix fold, found individually or in combination with other domains. The multiplicity of structural pattern enables it to form assemblies of diverse sizes, required for their abilities to confer multiple binding and structural roles of proteins. Three-dimensional structures of these repeats determined to date reveal a degree of structural variability that translates into the considerable functional versatility of this protein superfamily. Recent work on the ANK has proposed novel structural information, especially protein-lipid, protein-sugar and protein-protein interaction. Self-assembly of these repeats was also shown to prevent the associated protein in forming filaments. In this review, we summarize the latest findings and how the new structural information has increased our understanding of the structural determinants of ANK proteins. We discussed latest findings on how these proteins participate in various interactions to diversify the ANK roles in numerous biological processes, and explored the emerging and evolving field of designer ankyrins and its framework for protein engineering emphasizing on biotechnological applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

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

Dai, Shuyan; Sun, Cancan; Tan, Kemin

Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystalmore » structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.« less

Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs.

PubMed

Beyer, Andrea R; VieBrock, Lauren; Rodino, Kyle G; Miller, Daniel P; Tegels, Brittney K; Marconi, Richard T; Carlyon, Jason A

2015-10-01

A rising theme among intracellular microbes is the delivery of ankyrin repeat-containing effectors (Anks) that interact with target proteins to co-opt host cell functions. Orientia tsutsugamushi, an obligate intracellular bacterium and the etiologic agent of scrub typhus, encodes one of the largest Ank repertoires of any sequenced microorganism. They have been previously identified as type 1 secretion system substrates. Here, in silico and manual sequence analyses revealed that a large proportion of O. tsutsugamushi strain Ikeda Anks bear a eukaryotic/poxvirus-like F-box motif, which is known to recruit host cell SCF1 ubiquitin ligase machinery. We assessed the Anks for the ability to serve as F-box proteins. Coimmunoprecipitation assays demonstrated that F-box-containing Anks interact with overexpressed and/or endogenous SCF1 components. When coexpressed with FLAG-Ank4_01 or FLAG-Ank9, a glutathione S-transferase (GST)-tagged version of the SCF1 component SKP1 localized to subcellular sites of FLAG-Ank accumulation. The abilities of recombinant Anks to interact and colocalize with SKP1 were F-box dependent. GST-SKP1 precipitated O. tsutsugamushi-derived Ank9 from infected host cells, verifying both that the pathogen expresses Ank9 during infection and the protein's capability to bind SKP1. Aligning O. tsutsugamushi, poxviral, and eukaryotic F-box sequences delineated three F-box residues that are highly conserved and likely to be functionally important. Substitution of these residues ablated the ability of GFP-Ank9 to interact with GST-SKP1. These results demonstrate that O. tsutsugamushi strain Ikeda Anks can co-opt host cell polyubiquitination machinery, provide the first evidence that an O. tsutsugamushi Ank does so during infection, and advance overall understanding of microbial F-box proteins. Ankyrin repeat-containing proteins (Anks) are important virulence factors of intracellular bacteria that mediate protein-protein interactions with host cell targets

Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs

PubMed Central

Beyer, Andrea R.; VieBrock, Lauren; Rodino, Kyle G.; Miller, Daniel P.; Tegels, Brittney K.; Marconi, Richard T.

2015-01-01

ABSTRACT A rising theme among intracellular microbes is the delivery of ankyrin repeat-containing effectors (Anks) that interact with target proteins to co-opt host cell functions. Orientia tsutsugamushi, an obligate intracellular bacterium and the etiologic agent of scrub typhus, encodes one of the largest Ank repertoires of any sequenced microorganism. They have been previously identified as type 1 secretion system substrates. Here, in silico and manual sequence analyses revealed that a large proportion of O. tsutsugamushi strain Ikeda Anks bear a eukaryotic/poxvirus-like F-box motif, which is known to recruit host cell SCF1 ubiquitin ligase machinery. We assessed the Anks for the ability to serve as F-box proteins. Coimmunoprecipitation assays demonstrated that F-box-containing Anks interact with overexpressed and/or endogenous SCF1 components. When coexpressed with FLAG-Ank4_01 or FLAG-Ank9, a glutathione S-transferase (GST)-tagged version of the SCF1 component SKP1 localized to subcellular sites of FLAG-Ank accumulation. The abilities of recombinant Anks to interact and colocalize with SKP1 were F-box dependent. GST-SKP1 precipitated O. tsutsugamushi-derived Ank9 from infected host cells, verifying both that the pathogen expresses Ank9 during infection and the protein's capability to bind SKP1. Aligning O. tsutsugamushi, poxviral, and eukaryotic F-box sequences delineated three F-box residues that are highly conserved and likely to be functionally important. Substitution of these residues ablated the ability of GFP-Ank9 to interact with GST-SKP1. These results demonstrate that O. tsutsugamushi strain Ikeda Anks can co-opt host cell polyubiquitination machinery, provide the first evidence that an O. tsutsugamushi Ank does so during infection, and advance overall understanding of microbial F-box proteins. IMPORTANCE Ankyrin repeat-containing proteins (Anks) are important virulence factors of intracellular bacteria that mediate protein-protein interactions with

Solution properties of the archaeal CRISPR DNA repeat-binding homeodomain protein Cbp2

PubMed Central

Kenchappa, Chandra S.; Heidarsson, Pétur O.; Kragelund, Birthe B.; Garrett, Roger A.; Poulsen, Flemming M.

2013-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR) form the basis of diverse adaptive immune systems directed primarily against invading genetic elements of archaea and bacteria. Cbp1 of the crenarchaeal thermoacidophilic order Sulfolobales, carrying three imperfect repeats, binds specifically to CRISPR DNA repeats and has been implicated in facilitating production of long transcripts from CRISPR loci. Here, a second related class of CRISPR DNA repeat-binding protein, denoted Cbp2, is characterized that contains two imperfect repeats and is found amongst members of the crenarchaeal thermoneutrophilic order Desulfurococcales. DNA repeat-binding properties of the Hyperthermus butylicus protein Cbp2Hb were characterized and its three-dimensional structure was determined by NMR spectroscopy. The two repeats generate helix-turn-helix structures separated by a basic linker that is implicated in facilitating high affinity DNA binding of Cbp2 by tethering the two domains. Structural studies on mutant proteins provide support for Cys7 and Cys28 enhancing high thermal stability of Cbp2Hb through disulphide bridge formation. Consistent with their proposed CRISPR transcriptional regulatory role, Cbp2Hb and, by inference, other Cbp1 and Cbp2 proteins are closely related in structure to homeodomain proteins with linked helix-turn-helix (HTH) domains, in particular the paired domain Pax and Myb family proteins that are involved in eukaryal transcriptional regulation. PMID:23325851

A carrot leucine-rich-repeat protein that inhibits ice recrystallization.

PubMed

Worrall, D; Elias, L; Ashford, D; Smallwood, M; Sidebottom, C; Lillford, P; Telford, J; Holt, C; Bowles, D

1998-10-02

Many organisms adapted to live at subzero temperatures express antifreeze proteins that improve their tolerance to freezing. Although structurally diverse, all antifreeze proteins interact with ice surfaces, depress the freezing temperature of aqueous solutions, and inhibit ice crystal growth. A protein purified from carrot shares these functional features with antifreeze proteins of fish. Expression of the carrot complementary DNA in tobacco resulted in the accumulation of antifreeze activity in the apoplast of plants grown at greenhouse temperatures. The sequence of carrot antifreeze protein is similar to that of polygalacturonase inhibitor proteins and contains leucine-rich repeats.

RepeatsDB-lite: a web server for unit annotation of tandem repeat proteins.

PubMed

Hirsh, Layla; Paladin, Lisanna; Piovesan, Damiano; Tosatto, Silvio C E

2018-05-09

RepeatsDB-lite (http://protein.bio.unipd.it/repeatsdb-lite) is a web server for the prediction of repetitive structural elements and units in tandem repeat (TR) proteins. TRs are a widespread but poorly annotated class of non-globular proteins carrying heterogeneous functions. RepeatsDB-lite extends the prediction to all TR types and strongly improves the performance both in terms of computational time and accuracy over previous methods, with precision above 95% for solenoid structures. The algorithm exploits an improved TR unit library derived from the RepeatsDB database to perform an iterative structural search and assignment. The web interface provides tools for analyzing the evolutionary relationships between units and manually refine the prediction by changing unit positions and protein classification. An all-against-all structure-based sequence similarity matrix is calculated and visualized in real-time for every user edit. Reviewed predictions can be submitted to RepeatsDB for review and inclusion.

Upstream mononucleotide A-repeats play a cis-regulatory role in mammals through the DICER1 and Ago proteins.

PubMed

Aporntewan, Chatchawit; Pin-on, Piyapat; Chaiyaratana, Nachol; Pongpanich, Monnat; Boonyaratanakornkit, Viroj; Mutirangura, Apiwat

2013-10-01

A-repeats are the simplest form of tandem repeats and are found ubiquitously throughout genomes. These mononucleotide repeats have been widely believed to be non-functional 'junk' DNA. However, studies in yeasts suggest that A-repeats play crucial biological functions, and their role in humans remains largely unknown. Here, we showed a non-random pattern of distribution of sense A- and T-repeats within 20 kb around transcription start sites (TSSs) in the human genome. Different distributions of these repeats are observed upstream and downstream of TSSs. Sense A-repeats are enriched upstream, whereas sense T-repeats are enriched downstream of TSSs. This enrichment directly correlates with repeat size. Genes with different functions contain different lengths of repeats. In humans, tissue-specific genes are enriched for short repeats of <10 bp, whereas housekeeping genes are enriched for long repeats of ≥10 bp. We demonstrated that DICER1 and Argonaute proteins are required for the cis-regulatory role of A-repeats. Moreover, in the presence of a synthetic polymer that mimics an A-repeat, protein binding to A-repeats was blocked, resulting in a dramatic change in the expression of genes containing upstream A-repeats. Our findings suggest a length-dependent cis-regulatory function of A-repeats and that Argonaute proteins serve as trans-acting factors, binding to A-repeats.

Enhanced Expression of WD Repeat-Containing Protein 35 via Nuclear Factor-Kappa B Activation in Bupivacaine-Treated Neuro2a Cells

PubMed Central

Huang, Lei; Kondo, Fumio; Harato, Misako; Feng, Guo-Gang; Ishikawa, Naoshisa; Fujiwara, Yoshihiro; Okada, Shoshiro

2014-01-01

The family of WD repeat proteins comprises a large number of proteins and is involved in a wide variety of cellular processes such as signal transduction, cell growth, proliferation, and apoptosis. Bupivacaine is a sodium channel blocker administered for local infiltration, nerve block, epidural, and intrathecal anesthesia. Recently, we reported that bupivacaine induces reactive oxygen species (ROS) generation and p38 mitogen-activated protein kinase (MAPK) activation, resulting in an increase in the expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. It has been shown that ROS activate MAPK through phosphorylation, followed by activation of nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1). The present study was undertaken to test whether NF-κB and c-Jun/AP-1 are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Bupivacaine activated both NF-κB and c-Jun in Neuro2a cells. APDC, an NF-κB inhibitor, attenuated the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. GW9662, a selective peroxisome proliferator-activated receptor-γ antagonist, enhanced the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. In contrast, c-Jun siRNA did not inhibit the bupivacaine-induced increase in WDR35 mRNA expression. These results indicate that bupivacaine induces the activation of transcription factors NF-κB and c-Jun/AP-1 in Neuro2a cells, while activation of NF-κB is involved in bupivacaine-induced increases in WDR35 expression. PMID:24466034

Transfer buffer containing methanol can be reused multiple times in protein electrotransfer.

PubMed

Pettegrew, Colin J; Jayini, Renuka; Islam, M Rafiq

2009-04-01

We investigated the feasibility of repeated use of transfer buffer containing methanol in electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to polyvinylidene difluoride (PVDF) membrane using a prestained protein marker of broad molecular sizes. Transfer of the antitumor protein p53 in HEK293T cell extracts, using fresh and used transfer buffer, followed by detection with anti-p53 antibody was also performed to test detectability in immunoblot. Results from these experiments indicate that the transfer buffer can be reused at least five times and maintain a similar extent of protein transfer to PVDF membrane. Repeated use of the transfer buffer containing methanol will significantly reduce the volume of hazardous waste generated and its disposal cost as well as its adverse effect on environment.

Nucleotide-Binding Domain Leucine-Rich Repeat Containing Proteins and Intestinal Microbiota: Pivotal Players in Colitis and Colitis-Associated Cancer Development.

PubMed

Prossomariti, Anna; Sokol, Harry; Ricciardiello, Luigi

2018-01-01

The nucleotide-binding domain leucine-rich repeat containing (NLR) proteins play a fundamental role in innate immunity and intestinal tissue repair. A dysbiotic intestinal microbiota, developed as a consequence of alterations in NLR proteins, has recently emerged as a crucial hit for the development of ulcerative colitis (UC) and colitis-associated cancer (CAC). The concept of the existence of functional axes interconnecting bacteria with NLR proteins in a causal role in intestinal inflammation and CAC aroused a great interest for the potential development of preventive and therapeutic strategies against UC and CAC. However, the most recent scientific evidence, which highlights many confounding factors in studies based on microbiota characterization, underlines the need for an in-depth reconsideration of the data obtained until now. The purpose of this review is to discuss the recent findings concerning the cross talk between the NLR signaling and the intestinal microbiota in UC and CAC development, and to highlight the open issues that should be explored and addressed in future studies.

Sushi repeat-containing protein 1: a novel disease-associated molecule in cerebral amyloid angiopathy.

PubMed

Inoue, Yasuteru; Ueda, Mitsuharu; Tasaki, Masayoshi; Takeshima, Akari; Nagatoshi, Akihito; Masuda, Teruaki; Misumi, Yohei; Kosaka, Takayuki; Nomura, Toshiya; Mizukami, Mayumi; Matsumoto, Sayaka; Yamashita, Taro; Takahashi, Hitoshi; Kakita, Akiyoshi; Ando, Yukio

2017-10-01

Sporadic cerebral amyloid angiopathy (CAA) is characterized by cerebrovascular amyloid beta (Aβ) deposits and causes cerebral hemorrhage and dementia. The exact molecules that co-accumulate with cerebrovascular Aβ deposits are still not fully known. In our study here, we performed proteomic analyses with microdissected leptomeningeal arteries and cerebral neocortical arterioles from 8 cases with severe CAA, 12 cases with mild CAA, and 10 control cases without CAA, and we determined the levels of highly expressed proteins in cerebral blood vessels in CAA. We focused on sushi repeat-containing protein 1 (SRPX1), which is specifically expressed in CAA-affected cerebral blood vessels. Because SRPX1, which is known as a tumor suppressor gene, reportedly induced apoptosis in tumor cells, we hypothesized that SRPX1 may play an important role in Aβ-induced apoptosis in CAA. Immunohistochemical studies revealed that SRPX1 co-accumulated with Aβ deposits in cerebral blood vessels of all autopsied cases with severe CAA. In contrast, no SRPX1 co-accumulated with Aβ deposits in senile plaques. Furthermore, we demonstrated that both Aβ40 and Aβ42 bound to SRPX1 in vitro and enhanced SRPX1 expression in primary cultures of cerebrovascular smooth muscle cells. SRPX1 enhanced caspase activity induced by Aβ40. Knockdown of SRPX1, in contrast, reduced the formation of Aβ40 accumulations and the activity of caspase in cultured cerebrovascular smooth muscle cells. SRPX1 may thus be a novel molecule that is up-regulated in cerebrovascular Aβ deposits and that may increase Aβ-induced cerebrovascular degeneration in CAA.

Function of multiple Lis-Homology domain/WD-40 repeat-containing proteins in feed-forward transcriptional repression by silencing mediator for retinoic and thyroid receptor/nuclear receptor corepressor complexes.

PubMed

Choi, Hyo-Kyoung; Choi, Kyung-Chul; Kang, Hee-Bum; Kim, Han-Cheon; Lee, Yoo-Hyun; Haam, Seungjoo; Park, Hyoung-Gi; Yoon, Ho-Geun

2008-05-01

Lis-homology (LisH) motifs are involved in protein dimerization, and the discovery of the conserved N-terminal LisH domain in transducin beta-like protein 1 and its receptor (TBL1 and TBLR1) led us to examine the role of this domain in transcriptional repression. Here we show that multiple beta-transducin (WD-40) repeat-containing proteins interact to form oligomers in solution and that oligomerization depends on the presence of the LisH domain in each protein. Repression of transcription, as assayed using Gal4 fusion proteins, also depended on the presence of the LisH domain, suggesting that oligomerization is a prerequisite for efficient transcriptional repression. Furthermore, we show that the LisH domain is responsible for the binding to the hypoacetylated histone H4 tail and for stable chromatin targeting by the nuclear receptor corepressor complex. Mutations in conserved residues in the LisH motif of TBL1 and TBLR1 block histone binding, oligomerization, and transcriptional repression, supporting the functional importance of the LisH motif in transcriptional repression. Our results indicate that another WD-40 protein, TBL3, also preferentially binds to the N-terminal domain of TBL1 and TBLR1, and forms oligomers with other WD-40 proteins. Finally, we observed that the WD-40 proteins RbAp46 and RbAp48 of the sin3A corepressor complex failed to dimerize. We also found the specific interaction UbcH/E2 with TBL1, but not RbAp46/48. Altogether, our results thus indicate that the presence of multiple LisH/WD-40 repeat containing proteins is exclusive to nuclear receptor corepressor/ silencing mediator for retinoic and thyroid receptor complexes compared with other class 1 histone deacetylase-containing corepessor complexes.

[Collation between Za Liao of Qian Jin Yi Fang and Xin Xiu Ben Cao].

PubMed

Liang, Fei; Jiang, Li-Sheng; Li, Jian; Zhang, Wei; Zhang, Rui-Xian

2012-10-01

To analyze specific content of Za Liao of Qian Jin Yi Fang, this essay compares its content with related content of Xin Xiu Ben Cao, finding that Za Liao of Qian Jin Yi Fang is derived from the part of Jin An of Xin Xiu Ben Cao, which complements with herbal chapters from volume II to IV of Qian Jin Yi Fang. The texts in Za Liao can verify and collate part of Jin An, and thereby showing important literature value and great help for further studies on traditional Chinese medicines of Tang dynasty.

Upon Infection the Cellular WD Repeat-containing Protein 5 (WDR5) Localizes to Cytoplasmic Inclusion Bodies and Enhances Measles Virus Replication.

PubMed

Ma, Dzwokai; George, Cyril X; Nomburg, Jason; Pfaller, Christian K; Cattaneo, Roberto; Samuel, Charles E

2017-12-13

Replication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5 deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication. IMPORTANCE Measles virus is a human pathogen that remains a global concern with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that

StaRProtein, A Web Server for Prediction of the Stability of Repeat Proteins

PubMed Central

Xu, Yongtao; Zhou, Xu; Huang, Meilan

2015-01-01

Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins. PMID:25807112

Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins

DOE PAGES

Velásquez, André C.; Nomura, Kinya; Cooper, Max D.; ...

2017-04-19

The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein–protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies. Here, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs.more » HopM1-specific VLRBs (VLRM1) were expressed in planta in the cytosol, the trans-Golgi network, and the apoplast. Expression of VLRM1 was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLRM1 was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLRM1 specifically interacted in planta with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB. Later, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest in planta, with broad possibilities for their use by binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.« less

Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins

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

Velásquez, André C.; Nomura, Kinya; Cooper, Max D.

The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein–protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies. Here, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs.more » HopM1-specific VLRBs (VLRM1) were expressed in planta in the cytosol, the trans-Golgi network, and the apoplast. Expression of VLRM1 was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLRM1 was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLRM1 specifically interacted in planta with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB. Later, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest in planta, with broad possibilities for their use by binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.« less

Rational design of alpha-helical tandem repeat proteins with closed architectures

PubMed Central

Doyle, Lindsey; Hallinan, Jazmine; Bolduc, Jill; Parmeggiani, Fabio; Baker, David; Stoddard, Barry L.; Bradley, Philip

2015-01-01

Tandem repeat proteins, which are formed by repetition of modular units of protein sequence and structure, play important biological roles as macromolecular binding and scaffolding domains, enzymes, and building blocks for the assembly of fibrous materials1,2. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks3,4. The overall architecture of tandem repeat protein structures – which is dictated by the internal geometry and local packing of the repeat building blocks – is highly diverse, ranging from extended, super-helical folds that bind peptide, DNA, and RNA partners5–9, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis10. Here we report the development and validation of computational methods for de novo design of tandem repeat protein architectures driven purely by geometric criteria defining the inter-repeat geometry, without reference to the sequences and structures of existing repeat protein families. We have applied these methods to design a series of closed alpha-solenoid11 repeat structures (alpha-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the N- and C-termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering12–20, our design procedure does not rely on template sequence or structural information taken from natural repeat proteins and hence can produce structures unlike those seen in nature. As an example, we have successfully designed and validated closed alpha-solenoid repeats with a left-handed helical architecture that – to our knowledge – is not yet present in the protein structure database21. PMID:26675735

REPPER—repeats and their periodicities in fibrous proteins

PubMed Central

Gruber, Markus; Söding, Johannes; Lupas, Andrei N.

2005-01-01

REPPER (REPeats and their PERiodicities) is an integrated server that detects and analyzes regions with short gapless repeats in protein sequences or alignments. It finds periodicities by Fourier Transform (FTwin) and internal similarity analysis (REPwin). FTwin assigns numerical values to amino acids that reflect certain properties, for instance hydrophobicity, and gives information on corresponding periodicities. REPwin uses self-alignments and displays repeats that reveal significant internal similarities. Both programs use a sliding window to ensure that different periodic regions within the same protein are detected independently. FTwin and REPwin are complemented by secondary structure prediction (PSIPRED) and coiled coil prediction (COILS), making the server a versatile analysis tool for sequences of fibrous proteins. REPPER is available at . PMID:15980460

[Effects of land use structure on water quality in Xin'anjiang River].

PubMed

Cao, Fang-Fang; Li, Xue; Wang, Dong; Zhao, Yue; Wang, Yu-Qiu

2013-07-01

Take Xin'anjiang upstream watershed as a case study. Based on data of interpreting TM orthophoto images and water quality monitoring in May 2010, the land use map of Xin'anjiang River, which was categorized to cultivated land, forestland, grassland, water body, building site, was obtained. Using ArcGIS hydrological and spatial analysis function, Xin'anjiang River was divided into eight sub-watersheds, and its watershed land use structure was analyzed. The water quality parameters such as TN, TP, permanganate index, fecal coliform bacteria were monitored from Jan 2010 to Dec 2010. The relations between water quality and land use were analyzed. The results showed that TN and NH4(+) -N had a significant temporal variation: dry season > wet season > normal river flow period, but other parameters did not vary significantly. In the space, Yuliang and Pukou were the most serious pollution sites. Cultivated land, water body, building site had a positive impact on water quality parameters, while there were negative correlation between the forestland and grassland. Annually, cultivated land had the most significantly important effect on TN, NH4(+) -N and permanganate index, and grassland had the most significantly important effect on TP. Cultivated land had the most prominently important impact on water quality parameters in dry season and wet season. What's more, in the normal river flow, cultivated land, grassland and forestland had the most remarkably important influence on TN, TP and fecal coliform bacteria respectively.

Algorithm to find distant repeats in a single protein sequence

PubMed Central

Banerjee, Nirjhar; Sarani, Rangarajan; Ranjani, Chellamuthu Vasuki; Sowmiya, Govindaraj; Michael, Daliah; Balakrishnan, Narayanasamy; Sekar, Kanagaraj

2008-01-01

Distant repeats in protein sequence play an important role in various aspects of protein analysis. A keen analysis of the distant repeats would enable to establish a firm relation of the repeats with respect to their function and three-dimensional structure during the evolutionary process. Further, it enlightens the diversity of duplication during the evolution. To this end, an algorithm has been developed to find all distant repeats in a protein sequence. The scores from Point Accepted Mutation (PAM) matrix has been deployed for the identification of amino acid substitutions while detecting the distant repeats. Due to the biological importance of distant repeats, the proposed algorithm will be of importance to structural biologists, molecular biologists, biochemists and researchers involved in phylogenetic and evolutionary studies. PMID:19052663

Selection of specific protein binders for pre-defined targets from an optimized library of artificial helicoidal repeat proteins (alphaRep).

PubMed

Guellouz, Asma; Valerio-Lepiniec, Marie; Urvoas, Agathe; Chevrel, Anne; Graille, Marc; Fourati-Kammoun, Zaineb; Desmadril, Michel; van Tilbeurgh, Herman; Minard, Philippe

2013-01-01

We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a "filtration" procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×10(9) independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties.

The number of genes encoding repeat domain-containing proteins positively correlates with genome size in amoebal giant viruses

PubMed Central

Shukla, Avi; Chatterjee, Anirvan

2018-01-01

Abstract Curiously, in viruses, the virion volume appears to be predominantly driven by genome length rather than the number of proteins it encodes or geometric constraints. With their large genome and giant particle size, amoebal viruses (AVs) are ideally suited to study the relationship between genome and virion size and explore the role of genome plasticity in their evolutionary success. Different genomic regions of AVs exhibit distinct genealogies. Although the vertically transferred core genes and their functions are universally conserved across the nucleocytoplasmic large DNA virus (NCLDV) families and are essential for their replication, the horizontally acquired genes are variable across families and are lineage-specific. When compared with other giant virus families, we observed a near–linear increase in the number of genes encoding repeat domain-containing proteins (RDCPs) with the increase in the genome size of AVs. From what is known about the functions of RDCPs in bacteria and eukaryotes and their prevalence in the AV genomes, we envisage important roles for RDCPs in the life cycle of AVs, their genome expansion, and plasticity. This observation also supports the evolution of AVs from a smaller viral ancestor by the acquisition of diverse gene families from the environment including RDCPs that might have helped in host adaption. PMID:29308275

Deletion of internal structured repeats increases the stability of a leucine-rich repeat protein, YopM

PubMed Central

Barrick, Doug

2011-01-01

Mapping the stability distributions of proteins in their native folded states provides a critical link between structure, thermodynamics, and function. Linear repeat proteins have proven more amenable to this kind of mapping than globular proteins. C-terminal deletion studies of YopM, a large, linear leucine-rich repeat (LRR) protein, show that stability is distributed quite heterogeneously, yet a high level of cooperativity is maintained [1]. Key components of this distribution are three interfaces that strongly stabilize adjacent sequences, thereby maintaining structural integrity and promoting cooperativity. To better understand the distribution of interaction energy around these critical interfaces, we studied internal (rather than terminal) deletions of three LRRs in this region, including one of these stabilizing interfaces. Contrary to our expectation that deletion of structured repeats should be destabilizing, we find that internal deletion of folded repeats can actually stabilize the native state, suggesting that these repeats are destabilizing, although paradoxically, they are folded in the native state. We identified two residues within this destabilizing segment that deviate from the consensus sequence at a position that normally forms a stacked leucine ladder in the hydrophobic core. Replacement of these nonconsensus residues with leucine is stabilizing. This stability enhancement can be reproduced in the context of nonnative interfaces, but it requires an extended hydrophobic core. Our results demonstrate that different LRRs vary widely in their contribution to stability, and that this variation is context-dependent. These two factors are likely to determine the types of rearrangements that lead to folded, functional proteins, and in turn, are likely to restrict the pathways available for the evolution of linear repeat proteins. PMID:21764506

Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep)

PubMed Central

Chevrel, Anne; Graille, Marc; Fourati-Kammoun, Zaineb; Desmadril, Michel; van Tilbeurgh, Herman; Minard, Philippe

2013-01-01

We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a “filtration” procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×109 independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties. PMID:24014183

Recombinant small glutamine-rich tetratricopeptide repeat-containing protein of Leishmania infantum: Potential vaccine and diagnostic application against visceral leishmaniasis.

PubMed

Dias, Daniel S; Ribeiro, Patrícia A F; Martins, Vívian T; Lage, Daniela P; Portela, Áquila S B; Costa, Lourena E; Salles, Beatriz C S; Lima, Mariana P; Ramos, Fernanda F; Santos, Thaís T O; Caligiorne, Rachel B; Chávez-Fumagalli, Miguel A; Silveira, Julia A G; Magalhães-Soares, Danielle F; Gonçalves, Denise U; Oliveira, Jamil S; Roatt, Bruno M; Duarte, Mariana C; Menezes-Souza, Daniel; Silva, Eduardo S; Galdino, Alexsandro S; Machado-de-Ávila, Ricardo A; Teixeira, Antonio L; Coelho, Eduardo A F

2017-11-01

Different Leishmania proteins have been evaluated in order to find a potential vaccine candidate or diagnostic marker capable of providing long lasting protection against infection or helping to identify infected mammalian hosts, respectively. However, just few molecules have fulfilled all the requirements to be evaluated. In the current study, we evaluated the prophylactic and diagnostic value against visceral leishmaniasis (VL) of a small glutamine-rich tetratricopeptide repeat-containing (SGT) protein from Leishmania infantum species. In a first step, the immune response elicited by the immunization using the recombinant protein (rSGT) plus saponin was evaluated in BALB/c mice. Immunized animals had a low parasitism in all evaluated organs. They developed a specific Th1 immune response, which was based on protein-specific production of IFN-γ, IL-12 and GM-CSF, and a humoral response dominated by antibodies of the IgG2a isotype. Both CD4 + and CD8 + T cells contributed to the IFN-γ production, showing that both T cell subtypes contribute to the resistance against infection. Regarding its value as a diagnostic marker, rSGT showed maximum sensitivity and specificity to serologically identify L. infantum-infected dog and human sera. No cross-reactivity with sera from humans or dogs that had other diseases was found. Although further studies are necessary to validate these findings, data showed here suggest immunogenicity of rSGT and its protective effect against murine VL, as well as its potential for the serodiagnosis of human and canine VL. Copyright © 2017 Elsevier Ltd. All rights reserved.

An Ankyrin Repeat-Containing Protein, Characterized as a Ubiquitin Ligase, Is Closely Associated with Membrane-Enclosed Organelles and Required for Pollen Germination and Pollen Tube Growth in Lily1[W

PubMed Central

Huang, Jian; Chen, Feng; Del Casino, Cecilia; Autino, Antonella; Shen, Mouhua; Yuan, Shuai; Peng, Jia; Shi, Hexin; Wang, Chen; Cresti, Mauro; Li, Yiqin

2006-01-01

Exhibiting rapid polarized growth, the pollen tube delivers the male gametes into the ovule for fertilization in higher plants. To get an overall picture of gene expression during pollen germination and pollen tube growth, we profiled the transcription patterns of 1,536 pollen cDNAs from lily (Lilium longiflorum) by microarray. Among those that exhibited significant differential expression, a cDNA named lily ankyrin repeat-containing protein (LlANK) was thoroughly studied. The full-length LlANK cDNA sequence predicts a protein containing five tandem ankyrin repeats and a RING zinc-finger domain. The LlANK protein possesses ubiquitin ligase activity in vitro. RNA blots demonstrated that LlANK transcript is present in mature pollen and its level, interestingly contrary to most pollen mRNAs, up-regulated significantly during pollen germination and pollen tube growth. When fused with green fluorescent protein and transiently expressed in pollen, LlANK was found dominantly associated with membrane-enclosed organelles as well as the generative cell. Overexpression of LlANK, however, led to abnormal growth of the pollen tube. On the other hand, transient silencing of LlANK impaired pollen germination and tube growth. Taken together, these results showed that LlANK is a ubiquitin ligase associated with membrane-enclosed organelles and required for polarized pollen tube growth. PMID:16461387

Hybrid Sterility in Rice (Oryza sativa L.) Involves the Tetratricopeptide Repeat Domain Containing Protein.

PubMed

Yu, Yang; Zhao, Zhigang; Shi, Yanrong; Tian, Hua; Liu, Linglong; Bian, Xiaofeng; Xu, Yang; Zheng, Xiaoming; Gan, Lu; Shen, Yumin; Wang, Chaolong; Yu, Xiaowen; Wang, Chunming; Zhang, Xin; Guo, Xiuping; Wang, Jiulin; Ikehashi, Hiroshi; Jiang, Ling; Wan, Jianmin

2016-07-01

Intersubspecific hybrid sterility is a common form of reproductive isolation in rice (Oryza sativa L.), which significantly hampers the utilization of heterosis between indica and japonica varieties. Here, we elucidated the mechanism of S7, which specially causes Aus-japonica/indica hybrid female sterility, through cytological and genetic analysis, map-based cloning, and transformation experiments. Abnormal positioning of polar nuclei and smaller embryo sac were observed in F1 compared with male and female parents. Female gametes carrying S7(cp) and S7(i) were aborted in S7(ai)/S7(cp) and S7(ai)/S7(i), respectively, whereas they were normal in both N22 and Dular possessing a neutral allele, S7(n) S7 was fine mapped to a 139-kb region in the centromere region on chromosome 7, where the recombination was remarkably suppressed due to aggregation of retrotransposons. Among 16 putative open reading frames (ORFs) localized in the mapping region, ORF3 encoding a tetratricopeptide repeat domain containing protein was highly expressed in the pistil. Transformation experiments demonstrated that ORF3 is the candidate gene: downregulated expression of ORF3 restored spikelet fertility and eliminated absolutely preferential transmission of S7(ai) in heterozygote S7(ai)/S7(cp); sterility occurred in the transformants Cpslo17-S7(ai) Our results may provide implications for overcoming hybrid embryo sac sterility in intersubspecific hybrid rice and utilization of hybrid heterosis for cultivated rice improvement. Copyright © 2016 by the Genetics Society of America.

Reduced dislocation density in Ga xIn 1–xP compositionally graded buffer layers through engineered glide plane switch

DOE PAGES

Schulte, Kevin L.; France, Ryan M.; McMahon, William E.; ...

2016-11-17

In this work we develop control over dislocation glide dynamics in Ga xIn 1-xP compositionally graded buffer layers (CGBs) through control of CuPt ordering on the group-III sublattice. The ordered structure is metastable in the bulk, so any glissile dislocation that disrupts the ordered pattern will release stored energy, and experience an increased glide force. Here we show how this connection between atomic ordering and dislocation glide force can be exploited to control the threading dislocation density (TDD) in Ga xIn 1-xP CGBs. When ordered Ga xIn 1-xP is graded from the GaAs lattice constant to InP, the order parametermore » ..eta.. decreases as x decreases, and dislocation glide switches from one set of glide planes to the other. This glide plane switch (GPS) is accompanied by the nucleation of dislocations on the new glide plane, which typically leads to increased TDD. We develop control of the GPS position within a Ga xIn 1-xP CGB through manipulation of deposition temperature, surfactant concentration, and strain-grading rate. We demonstrate a two-stage Ga xIn 1-xP CGB from GaAs to InP with sufficiently low TDD for high performance devices, such as the 4-junction inverted metamorphic multi-junction solar cell, achieved through careful control the GPS position. Here, experimental results are analyzed within the context of a model that considers the force balance on dislocations on the two competing glide planes as a function of the degree of ordering.« less

A Long Terminal Repeat-Containing Retrotransposon of Schizosaccharomyces pombe Expresses a Gag-Like Protein That Assembles into Virus-Like Particles Which Mediate Reverse Transcription

PubMed Central

Teysset, Laure; Dang, Van-Dinh; Kim, Min Kyung; Levin, Henry L.

2003-01-01

The Tf1 element of Schizosaccharomyces pombe is a long terminal repeat-containing retrotransposon that encodes functional protease, reverse transcriptase, and integrase proteins. Although these proteins are known to be necessary for protein processing, reverse transcription, and integration, respectively, the function of the protein thought to be Gag has not been determined. We present here the first electron microscopy of Tf1 particles. We tested whether the putative Gag of Tf1 was required for particle formation, packaging of RNA, and reverse transcription. We generated deletions of 10 amino acids in each of the four hydrophilic domains of the protein and found that all four mutations reduced transposition activity. The N-terminal deletion removed a nuclear localization signal and inhibited nuclear import of the transposon. The two mutations in the center of Gag destabilized the protein and resulted in no virus-like particles. The C-terminal deletion caused a defect in RNA packaging and, as a result, low levels of cDNA. The electron microscopy of cells expressing a truncated Tf1 showed that Gag alone was sufficient for the formation of virus-like particles. Taken together, these results indicate that Tf1 encodes a Gag protein that is a functional equivalent of the Gag proteins of retroviruses. PMID:12692246

RNA-binding Protein Trinucleotide repeat-containing 6A Regulates the Formation of Circular RNA 0006916, with Important Functions in Lung Cancer Cells.

PubMed

Dai, Xin; Zhang, Nan; Cheng, Ying; Yang, Ti; Chen, Yingnan; Liu, Zhenzhong; Wang, Zhishan; Yang, Chengfeng; Jiang, Yiguo

2018-05-03

Circular RNAs (circRNAs) are widespread and diverse endogenous RNAs distinct from traditional linear RNAs, which may regulate gene expression in eukaryotes. However, the function of human circRNAs, including their potential role in lung cancer, remains largely unknown. We screened the circRNA circ0006916, which was evidently down-regulated in 16HBE-T cells (anti-benzopyrene-trans-7, 8-dihydrodiol-9, 10-epoxide-transformed human bronchial epithelial cells), and in A549 and H460 cell lines. Silencing of circ0006916, but not its parental gene homer scaffolding protein 1 (HOMER1), promoted cell proliferation via speeding up the cell cycle process rather than by inhibiting apoptosis; conversely, overexpression of circ0006916 had the opposite effect. Luciferase screening assay indicated that circ0006916 bound to miR-522-3p and inhibited pleckstrin homology domain and leucine rich repeat protein phosphatase 1 (PHLPP1) activity. We also explored the effect of the RNA-binding protein trinucleotide repeat-containing 6A (TNRC6A) on circ0006916 production. Circ0006916 expression was decreased after silencing TNRC6A. TNRC6A bound to the intron regions around the circRNA-forming exons of circ0006916, as shown by RNA immunoprecipitation assay combined with sequencing analysis. The association of circ0006916 with TNRC6A was further verified by RNA pull-down assays. We then constructed a carrier and confirmed that TNRC6A binding to the flanked intron region of circ0006916 was necessary for generation of circ0006916. These results demonstrate that TNRC6A regulates the biogenesis of the circRNA circ0006916, which has a regulatory role in cell growth.

Antibodies against the mono-methylated arginine-glycine repeat (MMA-RG) of the Epstein-Barr virus nuclear antigen 2 (EBNA2) identify potential cellular proteins targeted in viral transformation.

PubMed

Ayoubian, Hiresh; Fröhlich, Thomas; Pogodski, Dagmar; Flatley, Andrew; Kremmer, Elisabeth; Schepers, Aloys; Feederle, Regina; Arnold, Georg J; Grässer, Friedrich A

2017-08-01

The Epstein-Barr virus is a human herpes virus with oncogenic potential. The virus-encoded nuclear antigen 2 (EBNA2) is a key mediator of viral tumorigenesis. EBNA2 features an arginine-glycine (RG) repeat at amino acids (aa)339-354 that is essential for the transformation of lymphocytes and contains symmetrically (SDMA) and asymmetrically (ADMA) di-methylated arginine residues. The SDMA-modified EBNA2 binds the survival motor neuron protein (SMN), thus mimicking SMD3, a cellular SDMA-containing protein that interacts with SMN. Accordingly, a monoclonal antibody (mAb) specific for the SDMA-modified RG repeat of EBNA2 also binds to SMD3. With the novel mAb 19D4 we now show that EBNA2 contains mono-methylated arginine (MMA) residues within the RG repeat. Using 19D4, we immune-precipitated and analysed by mass spectrometry cellular proteins in EBV-transformed B-cells that feature MMA motifs that are similar to the one in EBNA2. Among the cellular proteins identified, we confirmed by immunoprecipitation and/or Western blot analyses Aly/REF, Coilin, DDX5, FXR1, HNRNPK, LSM4, MRE11, NRIP, nucleolin, PRPF8, RBM26, SMD1 (SNRDP1) and THRAP3 proteins that are either known to contain MMA residues or feature RG repeat sequences that probably serve as methylation substrates. The identified proteins are involved in splicing, tumorigenesis, transcriptional activation, DNA stability and RNA processing or export. Furthermore, we found that several proteins involved in energy metabolism are associated with MMA-modified proteins. Interestingly, the viral EBNA1 protein that features methylated RG repeat motifs also reacted with the antibodies. Our results indicate that the region between aa 34-52 of EBNA1 contains ADMA or SDMA residues, while the region between aa 328-377 mainly contains MMA residues.

Effects of Dao De Xin Xi Exercise on Balance and Quality of Life in Thai Elderly Women

PubMed Central

Intarakamhang, Patrawut; Chintanaprawasee, Pantipa

2012-01-01

The objective of this study was to evaluate the effects of a 12-week Dao De Xin Xi exercise, modified short forms of Tai Chi, on balance and quality of life in Thai elderly population. Quasi-Experimental research, pretest-posttest one group design was done at Physical Medicine and Rehabilitation Department, Phramongkutklao Hospital. Thai healthy elderly women over the age of 60, requiring regular Dao De Xin Xi exercise were recruited from either patients or workers in the hospital. A 60-minute Dao De Xin Xi exercise class was set as 3 times per week for 12 weeks. At baseline and 12 weeks, participants were tested in their static balance (Single-Leg Stance Timed Test with eyes open and close), dynamic balance (Expanded Timed Up and Go (ETUG) Test). Quality of life was measured by the abbreviated Thai version of the World Health Organization Quality of Life (WHOQOL-BREF) questionnaire. Fourteen females were studied with mean age of 62.8±4.3 years old. The Single-Leg Stance Timed Test with eyes open and close, Expanded Timed Up and Go (ETUG) Test improved significantly (before versus after exercises p <0.001). Significant improvement in quality of life were also found in each 4 domains, including physical health, psychological, social relationship, and environment (before versus after exercises p =0.001, 0.001, 0.004 and 0.005 respectively). No significant improvement was found only in the right Single-Leg Stance Timed Test with eyes close (p =0.091). A three times per week for 12-week Dao De Xin Xi exercise may help Thai elderly women improve both static, dynamic balance and quality of life. PMID:22980114

Effects of Dao De Xin Xi exercise on balance and quality of life in Thai elderly women.

PubMed

Intarakamhang, Patrawut; Chintanaprawasee, Pantipa

2011-12-29

The objective of this study was to evaluate the effects of a 12-week Dao De Xin Xi exercise, modified short forms of Tai Chi, on balance and quality of life in Thai elderly population. Quasi-Experimental research, pretest-posttest one group design was done at Physical Medicine and Rehabilitation Department, Phramongkutklao Hospital. Thai healthy elderly women over the age of 60, requiring regular Dao De Xin Xi exercise were recruited from either patients or workers in the hospital. A 60-minute Dao De Xin Xi exercise class was set as 3 times per week for 12 weeks. At baseline and 12 weeks, participants were tested in their static balance (Single-Leg Stance Timed Test with eyes open and close), dynamic balance (Expanded Timed Up and Go (ETUG) Test). Quality of life was measured by the abbreviated Thai version of the World Health Organization Quality of Life (WHOQOL-BREF) questionnaire. Fourteen females were studied with mean age of 62.8±4.3 years old. The Single-Leg Stance Timed Test with eyes open and close, Expanded Timed Up and Go (ETUG) Test improved significantly (before versus after exercises p <0.001). Significant improvement in quality of life were also found in each 4 domains, including physical health, psychological, social relationship, and environment (before versus after exercises p =0.001, 0.001, 0.004 and 0.005 respectively). No significant improvement was found only in the right Single-Leg Stance Timed Test with eyes close (p =0.091). A three times per week for 12-week Dao De Xin Xi exercise may help Thai elderly women improve both static, dynamic balance and quality of life.

Tong-xin-luo capsule for patients with coronary heart disease after percutaneous coronary intervention.

PubMed

Mao, Chen; Fu, Xiao-Hong; Yuan, Jin-Qiu; Yang, Zu-Yao; Chung, Vincent C H; Qin, Ying; Huang, Yafang; Tam, Wilson Wai San; Kwong, Joey S W; Xie, Wei; Tang, Jin-Ling

2015-05-21

Percutaneous coronary intervention (PCI) is a standard treatment for coronary heart disease (CHD). Restenosis, defined as a 50% reduction in luminal diameter at six months after PCI, indicates a need for revascularisation. Restenosis has proven to be a major drawback to PCI. Tong-xin-luo is one of the prophylactic strategies for cardiovascular events in patients after PCI that is widely used in China, but its efficacy and safety have not been systematically evaluated. To systematically assess the efficacy and safety of Tong-xin-luo capsules in preventing cardiovascular events after PCI in patients with CHD. We searched the Cochrane Central Register of Controlled Trials in The Cochrane Library, MEDLINE (OVID), EMBASE (OVID), WanFang, Chinese Biomedical Database, Chinese Medical Current Contents, and China National Knowledge Infrastructure from their inception to June 2014. We also searched other resources, including ongoing trials and research registries. We applied no language restrictions. Randomised controlled trials of participants with CHD after PCI were included. Participants in the intervention group received Tong-xin-luo capsules for at least three months. Two review authors independently extracted data and assessed the risk of bias. Any disagreements were resolved by discussion with a third review author. The primary outcomes included occurrence of angiographic restenosis and adverse events; the secondary outcomes included myocardial infarction, heart failure, angina, all cause mortality, mortality due to any cardiovascular event, use of revascularisation, patient acceptability, quality of life and cost-effectiveness. Dichotomous data were measured with risk ratios (RRs) with 95% confidence intervals (CIs). Sixteen studies involving 1063 participants were identified. The risk of bias for fifteen studies was high and along with imprecision and possible publication bias, this lowered our confidence in the results. There was low quality evidence that Tong

Thrombospondin Type-1 Repeat Domain-Containing Proteins Are Strongly Expressed in the Head Region of Hydra.

PubMed

Hamaguchi-Hamada, Kayoko; Kurumata-Shigeto, Mami; Minobe, Sumiko; Fukuoka, Nozomi; Sato, Manami; Matsufuji, Miyuki; Koizumi, Osamu; Hamada, Shun

2016-01-01

The head region of Hydra, the hypostome, is a key body part for developmental control and the nervous system. We herein examined genes specifically expressed in the head region of Hydra oligactis using suppression subtractive hybridization (SSH) cloning. A total of 1414 subtracted clones were sequenced and found to be derived from at least 540 different genes by BLASTN analyses. Approximately 25% of the subtracted clones had sequences encoding thrombospondin type-1 repeat (TSR) domains, and were derived from 17 genes. We identified 11 TSR domain-containing genes among the top 36 genes that were the most frequently detected in our SSH library. Whole-mount in situ hybridization analyses confirmed that at least 13 out of 17 TSR domain-containing genes were expressed in the hypostome of Hydra oligactis. The prominent expression of TSR domain-containing genes suggests that these genes play significant roles in the hypostome of Hydra oligactis.

[Family of ribosomal proteins S1 contains unique conservative domain].

PubMed

Deriusheva, E I; Machulin, A V; Selivanova, O M; Serdiuk, I N

2010-01-01

Different representatives of bacteria have different number of amino acid residues in the ribosomal proteins S1. This number varies from 111 (Spiroplasma kunkelii) to 863 a.a. (Treponema pallidum). Traditionally and for lack of this protein three-dimensional structure, its architecture is represented as repeating S1 domains. Number of these domains depends on the protein's length. Domain's quantity and its boundaries data are contained in the specialized databases, such as SMART, Pfam and PROSITE. However, for the same object these data may be very different. For search of domain's quantity and its boundaries, new approach, based on the analysis of dicted secondary structure (PsiPred), was used. This approach allowed us to reveal structural domains in amino acid sequences of S1 proteins and at that number varied from one to six. Alignment of S1 proteins, containing different domain's number, with the S1 RNAbinding domain of Escherichia coli PNPase elicited a fact that in family of ribosomal proteins SI one domain has maximal homology with S1 domain from PNPase. This conservative domain migrates along polypeptide chain and locates in proteins, containing different domain's number, according to specified pattern. In this domain as well in the S1 domain from PNPase, residues Phe-19, Phe-22, His-34, Asp-64 and Arg-68 are clustered on the surface and formed RNA binding site.

A designed repeat protein as an affinity capture reagent

PubMed Central

Speltz, Elizabeth B.; Brown, Rebecca S.H.; Hajare, Holly S.; Schlieker, Christian; Regan, Lynne

2017-01-01

Repeat proteins are an attractive target for protein engineering and design. We have focused our attention on the design and engineering of one particular class - tetratricopeptide repeat (TPR) proteins. In previous work we have shown that the structure and stability of TPR proteins can be manipulated in a rational fashion [Cortajarena 2011; Main 2003]. Building on those studies, we have designed and characterized a number of different peptide-binding TPR modules and we have also assembled these modules into supramolecular arrays [Cortajarena 2009; Cortajarena 2008; Jackrel 2009; Kajander 2007]. Here we focus on the development of one such TPR-peptide interaction for a practical application – affinity purification. We illustrate the general utility of our designed protein interaction. Furthermore, this example highlights how basic research on protein-peptide interactions can lead to the development of novel reagents with important practical applications. PMID:26517897

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

PubMed

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

2012-11-07

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

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

PubMed Central

2012-01-01

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

Design of tryptophan-containing mutants of the symmetrical Pizza protein for biophysical studies.

PubMed

Noguchi, Hiroki; Mylemans, Bram; De Zitter, Elke; Van Meervelt, Luc; Tame, Jeremy R H; Voet, Arnout

2018-03-18

β-propeller proteins are highly symmetrical, being composed of a repeated motif with four anti-parallel β-sheets arranged around a central axis. Recently we designed the first completely symmetrical β-propeller protein, Pizza6, consisting of six identical tandem repeats. Pizza6 is expected to prove a useful building block for bionanotechnology, and also a tool to investigate the folding and evolution of β-propeller proteins. Folding studies are made difficult by the high stability and the lack of buried Trp residues to act as monitor fluorophores, so we have designed and characterized several Trp-containing Pizza6 derivatives. In total four proteins were designed, of which three could be purified and characterized. Crystal structures confirm these mutant proteins maintain the expected structure, and a clear redshift of Trp fluorescence emission could be observed upon denaturation. Among the derivative proteins, Pizza6-AYW appears to be the most suitable model protein for future folding/unfolding kinetics studies as it has a comparable stability as natural β-propeller proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Thrombospondin Type-1 Repeat Domain-Containing Proteins Are Strongly Expressed in the Head Region of Hydra

PubMed Central

Hamaguchi-Hamada, Kayoko; Kurumata-Shigeto, Mami; Minobe, Sumiko; Fukuoka, Nozomi; Sato, Manami; Matsufuji, Miyuki; Koizumi, Osamu; Hamada, Shun

2016-01-01

The head region of Hydra, the hypostome, is a key body part for developmental control and the nervous system. We herein examined genes specifically expressed in the head region of Hydra oligactis using suppression subtractive hybridization (SSH) cloning. A total of 1414 subtracted clones were sequenced and found to be derived from at least 540 different genes by BLASTN analyses. Approximately 25% of the subtracted clones had sequences encoding thrombospondin type-1 repeat (TSR) domains, and were derived from 17 genes. We identified 11 TSR domain-containing genes among the top 36 genes that were the most frequently detected in our SSH library. Whole-mount in situ hybridization analyses confirmed that at least 13 out of 17 TSR domain-containing genes were expressed in the hypostome of Hydra oligactis. The prominent expression of TSR domain-containing genes suggests that these genes play significant roles in the hypostome of Hydra oligactis. PMID:27043211

Design, production and molecular structure of a new family of artificial alpha-helicoidal repeat proteins (αRep) based on thermostable HEAT-like repeats.

PubMed

Urvoas, Agathe; Guellouz, Asma; Valerio-Lepiniec, Marie; Graille, Marc; Durand, Dominique; Desravines, Danielle C; van Tilbeurgh, Herman; Desmadril, Michel; Minard, Philippe

2010-11-26

Repeat proteins have a modular organization and a regular architecture that make them attractive models for design and directed evolution experiments. HEAT repeat proteins, although very common, have not been used as a scaffold for artificial proteins, probably because they are made of long and irregular repeats. Here, we present and validate a consensus sequence for artificial HEAT repeat proteins. The sequence was defined from the structure-based sequence analysis of a thermostable HEAT-like repeat protein. Appropriate sequences were identified for the N- and C-caps. A library of genes coding for artificial proteins based on this sequence design, named αRep, was assembled using new and versatile methodology based on circular amplification. Proteins picked randomly from this library are expressed as soluble proteins. The biophysical properties of proteins with different numbers of repeats and different combinations of side chains in hypervariable positions were characterized. Circular dichroism and differential scanning calorimetry experiments showed that all these proteins are folded cooperatively and are very stable (T(m) >70 °C). Stability of these proteins increases with the number of repeats. Detailed gel filtration and small-angle X-ray scattering studies showed that the purified proteins form either monomers or dimers. The X-ray structure of a stable dimeric variant structure was solved. The protein is folded with a highly regular topology and the repeat structure is organized, as expected, as pairs of alpha helices. In this protein variant, the dimerization interface results directly from the variable surface enriched in aromatic residues located in the randomized positions of the repeats. The dimer was crystallized both in an apo and in a PEG-bound form, revealing a very well defined binding crevice and some structure flexibility at the interface. This fortuitous binding site could later prove to be a useful binding site for other low molecular mass

Enhanced Expression of WD Repeat-Containing Protein 35 via CaMKK/AMPK Activation in Bupivacaine-Treated Neuro2a Cells

PubMed Central

Huang, Lei; Kondo, Fumio; Gosho, Masahiko; Feng, Guo-Gang; Harato, Misako; Xia, Zhong-yuan; Ishikawa, Naohisa; Fujiwara, Yoshihiro; Okada, Shoshiro

2014-01-01

We previously reported that bupivacaine induces reactive oxygen species (ROS) generation, p38 mitogen-activated protein kinase (MAPK) activation and nuclear factor-kappa B activation, resulting in an increase in expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. However, the identity of signaling upstream of p38 MAPK pathways to WDR35 expression remains unclear. It has been shown that AMP-activated protein kinase (AMPK) can activate p38 MAPK through diverse mechanisms. In addition, several kinases acting upstream of AMPK have been identified including Ca2+/calmodulin-dependent protein kinase kinase (CaMKK). Recent studies reported that AMPK may be involved in bupivacaine-induced cytotoxicity in Schwann cells and in human neuroblastoma SH-SY5Y cells. The present study was undertaken to test whether CaMKK and AMPK are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Our results showed that bupivacaine induced activation of AMPK and p38 MAPK in Neuro2a cells. The AMPK inhibitors, compound C and iodotubercidin, attenuated the bupivacaine-induced activation of AMPK and p38 MAPK, resulting in an inhibition of the bupivacaine-induced increase in WDR35 expression. Treatment with the CaMKK inhibitor STO-609 also attenuated the bupivacaine-induced activation of AMPK and p38 MAPK, resulting in an inhibition of the bupivacaine-induced increase in WDR35 expression. These results suggest that bupivacaine activates AMPK and p38 MAPK via CaMKK in Neuro2a cells, and that the CaMKK/AMPK/p38 MAPK pathway is involved in regulating WDR35 expression. PMID:24859235

Seasonal-Spatial Distribution and Long-Term Variation of Transparency in Xin'anjiang Reservoir: Implications for Reservoir Management.

PubMed

Wu, Zhixu; Zhang, Yunlin; Zhou, Yongqiang; Liu, Mingliang; Shi, Kun; Yu, Zuoming

2015-08-12

Water transparency is a useful indicator of water quality or productivity and is widely used to detect long-term changes in the water quality and eutrophication of lake ecosystems. Based on short-term spatial observations in the spring, summer, and winter and on long-term site-specific observation from 1988 to 2013, the spatial, seasonal, long-term variations, and the factors affecting transparency are presented for Xin'anjiang Reservoir (China). Spatially, transparency was high in the open water but low in the bays and the inflowing river mouths, reflecting the effect of river runoff. The seasonal effects were distinct, with lower values in the summer than in the winter, most likely due to river runoff and phytoplankton biomass increases. The transparency decreased significantly with a linear slope of 0.079 m/year, indicating a 2.05 m decrease and a marked decrease in water quality. A marked increase occurred in chlorophyll a (Chla) concentration, and a significant correlation was found between the transparency and Chla concentration, indicating that phytoplankton biomass can partially explain the long-term trend of transparency in Xin'anjiang Reservoir. The river input and phytoplankton biomass increase were associated with soil erosion and nutrient loss in the catchment. Our study will support future management of water quality in Xin'anjiang Reservoir.

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

NASA Technical Reports Server (NTRS)

Safadi, F.; Reddy, V. S.; Reddy, A. S.

2000-01-01

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals.

PubMed

Zlopasa, Livija; Brachner, Andreas; Foisner, Roland

2016-06-01

Ankyrin repeats and LEM domain containing protein 1 (Ankle1) belongs to the LEM protein family, whose members share a chromatin-interacting LEM motif. Unlike most other LEM proteins, Ankle1 is not an integral protein of the inner nuclear membrane but shuttles between the nucleus and the cytoplasm. It contains a GIY-YIG-type nuclease domain, but its function is unknown. The mammalian genome encodes only one other GIY-YIG domain protein, termed Slx1. Slx1 has been described as a resolvase that processes Holliday junctions during homologous recombination-mediated DNA double strand break repair. Resolvase activity is regulated in a spatial and temporal manner during the cell cycle. We hypothesized that Ankle1 may have a similar function and its nucleo-cytoplasmic shuttling may contribute to the regulation of Ankle1 activity. Hence, we aimed at identifying the domains mediating Ankle1 shuttling and investigating whether cellular localization is affected during DNA damage response. Sequence analysis predicts the presence of two canonical nuclear import and export signals in Ankle1. Immunofluorescence microscopy of cells expressing wild-type and various mutated Ankle1-fusion proteins revealed a C-terminally located classical monopartite nuclear localization signal and a centrally located CRM1-dependent nuclear export signal that mediate nucleo-cytoplasmic shuttling of Ankle1. These sequences are also functional in heterologous proteins. The predominant localization of Ankle1 in the cytoplasm, however, does not change upon induction of several DNA damage response pathways throughout the cell cycle. We identified the domains mediating nuclear import and export of Ankle1. Ankle1's cellular localization was not affected following DNA damage.

Chlorovirus Skp1-binding ankyrin repeat protein interplay and mimicry of cellular ubiquitin ligase machinery.

PubMed

Noel, Eric A; Kang, Ming; Adamec, Jiri; Van Etten, James L; Oyler, George A

2014-12-01

The ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. Members of the genus Chlorovirus are proposed to share an ancestral lineage with a broader group of related viruses, nucleo-cytoplasmic large DNA viruses (NCLDV). Chloroviruses encode an Skp1 homolog and ankyrin repeat (ANK) proteins. Several chlorovirus-encoded ANK repeats contain C-terminal domains characteristic of cellular F-boxes or related NCLDV chordopox PRANC (pox protein repeats of ankyrin at C-terminal) domains. These observations suggested that this unique combination of Skp1 and ANK repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic chlorovirus Paramecium bursaria chlorella virus-1 (PBCV-1) that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned similarly to cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three chlorovirus types. Collectively, our results indicate that chloroviruses have evolved complementing Skp1 and ANK proteins that mimic cellular SCF-associated proteins. Viruses have evolved ways to direct ubiquitination events in order to create environments conducive to their replication. As

Direct Observation of Parallel Folding Pathways Revealed Using a Symmetric Repeat Protein System

PubMed Central

Aksel, Tural; Barrick, Doug

2014-01-01

Although progress has been made to determine the native fold of a polypeptide from its primary structure, the diversity of pathways that connect the unfolded and folded states has not been adequately explored. Theoretical and computational studies predict that proteins fold through parallel pathways on funneled energy landscapes, although experimental detection of pathway diversity has been challenging. Here, we exploit the high translational symmetry and the direct length variation afforded by linear repeat proteins to directly detect folding through parallel pathways. By comparing folding rates of consensus ankyrin repeat proteins (CARPs), we find a clear increase in folding rates with increasing size and repeat number, although the size of the transition states (estimated from denaturant sensitivity) remains unchanged. The increase in folding rate with chain length, as opposed to a decrease expected from typical models for globular proteins, is a clear demonstration of parallel pathways. This conclusion is not dependent on extensive curve-fitting or structural perturbation of protein structure. By globally fitting a simple parallel-Ising pathway model, we have directly measured nucleation and propagation rates in protein folding, and have quantified the fluxes along each path, providing a detailed energy landscape for folding. This finding of parallel pathways differs from results from kinetic studies of repeat-proteins composed of sequence-variable repeats, where modest repeat-to-repeat energy variation coalesces folding into a single, dominant channel. Thus, for globular proteins, which have much higher variation in local structure and topology, parallel pathways are expected to be the exception rather than the rule. PMID:24988356

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

PubMed

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

2004-06-23

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

Origin of a folded repeat protein from an intrinsically disordered ancestor

PubMed Central

Zhu, Hongbo; Sepulveda, Edgardo; Hartmann, Marcus D; Kogenaru, Manjunatha; Ursinus, Astrid; Sulz, Eva; Albrecht, Reinhard; Coles, Murray; Martin, Jörg; Lupas, Andrei N

2016-01-01

Repetitive proteins are thought to have arisen through the amplification of subdomain-sized peptides. Many of these originated in a non-repetitive context as cofactors of RNA-based replication and catalysis, and required the RNA to assume their active conformation. In search of the origins of one of the most widespread repeat protein families, the tetratricopeptide repeat (TPR), we identified several potential homologs of its repeated helical hairpin in non-repetitive proteins, including the putatively ancient ribosomal protein S20 (RPS20), which only becomes structured in the context of the ribosome. We evaluated the ability of the RPS20 hairpin to form a TPR fold by amplification and obtained structures identical to natural TPRs for variants with 2–5 point mutations per repeat. The mutations were neutral in the parent organism, suggesting that they could have been sampled in the course of evolution. TPRs could thus have plausibly arisen by amplification from an ancestral helical hairpin. DOI: http://dx.doi.org/10.7554/eLife.16761.001 PMID:27623012

Evidence that a proposed repeated segment of glutamine residues is expressed in the Huntington disease protein

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

Jou, Y.S.; Myers, R.M.

1994-09-01

Huntington disease (HD) appears to be caused by a mutation that results in an expanded number of CAG repeats at the 5{prime} end of the gene. The nucleotide sequence of the gene and cDNA clones predicts a 347 kd protein that contains a stretch of polyglutamine, encoded by the CAG repeat, located 17 amino acids downstream from the proposed translation initiation site. Because understanding the mechanisms of the pathology of HD depends on whether the CAG-repeat is expressed in the protein, we used antibodies directed against portions of the predicted HD gene product to probe the structure of the proteinmore » in tissue culture cells. Two peptides, one located amino-terminal to the proposed polyglutamine stretch (hd1 peptide FESLKSFQQ from amino acids 11-19) and one located in the carboxy-terminal half of the predicted protein (hd2 peptide QQPRNKPLK from amino acids 2531-2539), were used to elicit polyclonal antibodies in NZW rabbits. We affinity-purified the antibodies and used them to analyze the HD protein. Both antisera specifically recognize the peptides used to elicit them, as well as the appropriate portions of the HD protein expressed in E. coli. Western blot analysis showed that both antisera recognize a protein with an apparent molecular weight of approximately 350,000 in human, monkey, rat and mouse cell lines, including two neutronal cell lines. These results, in combination with immunoprecipitation experiments, suggest strongly that the proposed polyglutamine stretch is indeed translated in the HD protein and is evolutionarily conserved in various mammalian species.« less

Tandem Repeat Proteins Inspired By Squid Ring Teeth

NASA Astrophysics Data System (ADS)

Pena-Francesch, Abdon

Proteins are large biomolecules consisting of long chains of amino acids that hierarchically assemble into complex structures, and provide a variety of building blocks for biological materials. The repetition of structural building blocks is a natural evolutionary strategy for increasing the complexity and stability of protein structures. However, the relationship between amino acid sequence, structure, and material properties of protein systems remains unclear due to the lack of control over the protein sequence and the intricacies of the assembly process. In order to investigate the repetition of protein building blocks, a recently discovered protein from squids is examined as an ideal protein system. Squid ring teeth are predatory appendages located inside the suction cups that provide a strong grasp of prey, and are solely composed of a group of proteins with tandem repetition of building blocks. The objective of this thesis is the understanding of sequence, structure and property relationship in repetitive protein materials inspired in squid ring teeth for the first time. Specifically, this work focuses on squid-inspired structural proteins with tandem repeat units in their sequence (i.e., repetition of alternating building blocks) that are physically cross-linked via beta-sheet structures. The research work presented here tests the hypothesis that, in these systems, increasing the number of building blocks in the polypeptide chain decreases the protein network defects and improves the material properties. Hence, the sequence, nanostructure, and properties (thermal, mechanical, and conducting) of tandem repeat squid-inspired protein materials are examined. Spectroscopic structural analysis, advanced materials characterization, and entropic elasticity theory are combined to elucidate the structure and material properties of these repetitive proteins. This approach is applied not only to native squid proteins but also to squid-inspired synthetic polypeptides

Immunogenicity of a Prime-Boost Vaccine Containing the Circumsporozoite Proteins of Plasmodium vivax in Rodents

PubMed Central

Teixeira, Lais H.; Tararam, Cibele A.; Lasaro, Marcio O.; Camacho, Ariane G. A.; Ersching, Jonatan; Leal, Monica T.; Herrera, Sócrates; Bruna-Romero, Oscar; Soares, Irene S.; Nussenzweig, Ruth S.; Ertl, Hildegund C. J.; Nussenzweig, Victor

2014-01-01

Plasmodium vivax is the most widespread and the second most prevalent malaria-causing species in the world. Current measures used to control the transmission of this disease would benefit from the development of an efficacious vaccine. In the case of the deadly parasite P. falciparum, the recombinant RTS,S vaccine containing the circumsporozoite antigen (CSP) consistently protects 30 to 50% of human volunteers against infection and is undergoing phase III clinical trials in Africa with similar efficacy. These findings encouraged us to develop a P. vivax vaccine containing the three circulating allelic forms of P. vivax CSP. Toward this goal, we generated three recombinant bacterial proteins representing the CSP alleles, as well as a hybrid polypeptide called PvCSP-All-CSP-epitopes. This hybrid contains the conserved N and C termini of P. vivax CSP and the three variant repeat domains in tandem. We also generated simian and human recombinant replication-defective adenovirus vectors expressing PvCSP-All-CSP-epitopes. Mice immunized with the mixture of recombinant proteins in a formulation containing the adjuvant poly(I·C) developed high and long-lasting serum IgG titers comparable to those elicited by proteins emulsified in complete Freund's adjuvant. Antibody titers were similar in mice immunized with homologous (protein-protein) and heterologous (adenovirus-protein) vaccine regimens. The antibodies recognized the three allelic forms of CSP, reacted to the repeated and nonrepeated regions of CSP, and recognized sporozoites expressing the alleles VK210 and VK247. The vaccine formulations described in this work should be useful for the further development of an anti-P. vivax vaccine. PMID:24478093

[Diversity of ectomycorrhizal fungi associated with Picea asperata in Xin Jiashan Forest of Qinling Mountains].

PubMed

Geng, Rong; Geng, Zengchao; Huang, Jian; He, Wenxiang; Hou, Lin; She, Diao; Zhao, Jun; Shang, Jie

2015-07-04

To study the diversity of ectomycorrhizal fungi associated with Picea asperata in Xin Jiashan Forest of Qinling Mountains. This method combined the field investigation, morphological and molecular biology to identify ectomycorrhizal fungi. There were 37 different ectomycorrhizal fungi under 14 genera of 10 families on spruce in Xin Jiashan Forest of Qinling Mountains, 34 types belonged to Basidiomycetes, 1 to Ascomycete and 2 to unknown species. Among these identified ectomycorrhizal fungi types, Inocybe sp. was the dominant group; Russula nauseosa was the most dominant species; Hygrophorus sp., Tomentella coerulea, Inocybe sp. 1, Helotiaceae sp. and Lactarius deterrimus were common species; and the rest species were rare species. The large number but relatively rare species of dominant family and the small number but relatively more species of rare family survived in ectomycorrhizal fungal communities of Picea asperata. For the extreme degradation in arid area of western ecological system, identifing some rare family for further development and utilization had very important practical significance.

Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins.

PubMed

Kramer, Marianne C; Liang, Dongming; Tatomer, Deirdre C; Gold, Beth; March, Zachary M; Cherry, Sara; Wilusz, Jeremy E

2015-10-15

Thousands of eukaryotic protein-coding genes are noncanonically spliced to produce circular RNAs. Bioinformatics has indicated that long introns generally flank exons that circularize in Drosophila, but the underlying mechanisms by which these circular RNAs are generated are largely unknown. Here, using extensive mutagenesis of expression plasmids and RNAi screening, we reveal that circularization of the Drosophila laccase2 gene is regulated by both intronic repeats and trans-acting splicing factors. Analogous to what has been observed in humans and mice, base-pairing between highly complementary transposable elements facilitates backsplicing. Long flanking repeats (∼ 400 nucleotides [nt]) promote circularization cotranscriptionally, whereas pre-mRNAs containing minimal repeats (<40 nt) generate circular RNAs predominately after 3' end processing. Unlike the previously characterized Muscleblind (Mbl) circular RNA, which requires the Mbl protein for its biogenesis, we found that Laccase2 circular RNA levels are not controlled by Mbl or the Laccase2 gene product but rather by multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine-arginine) proteins acting in a combinatorial manner. hnRNP and SR proteins also regulate the expression of other Drosophila circular RNAs, including Plexin A (PlexA), suggesting a common strategy for regulating backsplicing. Furthermore, the laccase2 flanking introns support efficient circularization of diverse exons in Drosophila and human cells, providing a new tool for exploring the functional consequences of circular RNA expression across eukaryotes. © 2015 Kramer et al.; Published by Cold Spring Harbor Laboratory Press.

Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins

PubMed Central

Kramer, Marianne C.; Liang, Dongming; Tatomer, Deirdre C.; Gold, Beth; March, Zachary M.; Cherry, Sara; Wilusz, Jeremy E.

2015-01-01

Thousands of eukaryotic protein-coding genes are noncanonically spliced to produce circular RNAs. Bioinformatics has indicated that long introns generally flank exons that circularize in Drosophila, but the underlying mechanisms by which these circular RNAs are generated are largely unknown. Here, using extensive mutagenesis of expression plasmids and RNAi screening, we reveal that circularization of the Drosophila laccase2 gene is regulated by both intronic repeats and trans-acting splicing factors. Analogous to what has been observed in humans and mice, base-pairing between highly complementary transposable elements facilitates backsplicing. Long flanking repeats (∼400 nucleotides [nt]) promote circularization cotranscriptionally, whereas pre-mRNAs containing minimal repeats (<40 nt) generate circular RNAs predominately after 3′ end processing. Unlike the previously characterized Muscleblind (Mbl) circular RNA, which requires the Mbl protein for its biogenesis, we found that Laccase2 circular RNA levels are not controlled by Mbl or the Laccase2 gene product but rather by multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine–arginine) proteins acting in a combinatorial manner. hnRNP and SR proteins also regulate the expression of other Drosophila circular RNAs, including Plexin A (PlexA), suggesting a common strategy for regulating backsplicing. Furthermore, the laccase2 flanking introns support efficient circularization of diverse exons in Drosophila and human cells, providing a new tool for exploring the functional consequences of circular RNA expression across eukaryotes. PMID:26450910

Multiple intermediates on the energy landscape of a 15-HEAT-repeat protein

PubMed Central

Tsytlonok, Maksym; Craig, Patricio O.; Sivertsson, Elin; Serquera, David; Perrett, Sarah; Best, Robert B.; Wolynes, Peter G.; Itzhaki, Laura S.

2014-01-01

Repeat proteins are a special class of modular, non-globular proteins composed of small structural motifs arrayed to form elongated architectures and stabilised solely by short-range contacts. We find a remarkable complexity in the unfolding of the large HEAT repeat protein PR65/A. In contrast to what has been seen for small repeat proteins in which unfolding propagates from one end, the HEAT array of PR65/A ruptures at multiple distant sites, leading to intermediate states with non-contiguous folded subdomains. Kinetic analysis allows us to define a network of intermediates and to delineate the pathways that connect them. There is a dominant sequence of unfolding, reflecting a non-uniform distribution of stability across the repeat array; however the unfolding of certain intermediates is competitive, leading to parallel pathways. Theoretical models accounting for the heterogeneous contact density in the folded structure are able to rationalize the variation in stability across the array. This variation in stability also suggests how folding may direct function in a large repeat protein: The stability distribution enables certain regions to present rigid motifs for molecular recognition while affording others flexibility to broaden the search area as in a fly-casting mechanism. Thus PR65/A uses the two ends of the repeat array to bind diverse partners and thereby coordinate the dephosphorylation of many different substrates and of multiple sites within hyperphosphorylated substrates. PMID:24120762

Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I

PubMed Central

Anand, Arvind; LeDoyt, Morgan; Karanian, Carson; Luthra, Amit; Koszelak-Rosenblum, Mary; Malkowski, Michael G.; Puthenveetil, Robbins; Vinogradova, Olga; Radolf, Justin D.

2015-01-01

We previously identified Treponema pallidum repeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprCN and TprCC) orthologous to regions in the major outer sheath protein (MOSPN and MOSPC) of Treponema denticola and that TprCC is solely responsible for β-barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSPC-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSPN-like domains are tethered within the periplasm. TprF, which does not contain a MOSPC-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSPN and MOSPC-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSPN-like domains enhance the structural integrity of the cell envelope by anchoring the β-barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP. PMID:25805501

In Vitro Expansion of CAG, CAA, and Mixed CAG/CAA Repeats.

PubMed

Figura, Grzegorz; Koscianska, Edyta; Krzyzosiak, Wlodzimierz J

2015-08-11

Polyglutamine diseases, including Huntington's disease and a number of spinocerebellar ataxias, are caused by expanded CAG repeats that are located in translated sequences of individual, functionally-unrelated genes. Only mutant proteins containing polyglutamine expansions have long been thought to be pathogenic, but recent evidence has implicated mutant transcripts containing long CAG repeats in pathogenic processes. The presence of two pathogenic factors prompted us to attempt to distinguish the effects triggered by mutant protein from those caused by mutant RNA in cellular models of polyglutamine diseases. We used the SLIP (Synthesis of Long Iterative Polynucleotide) method to generate plasmids expressing long CAG repeats (forming a hairpin structure), CAA-interrupted CAG repeats (forming multiple unstable hairpins) or pure CAA repeats (not forming any secondary structure). We successfully modified the original SLIP protocol to generate repeats of desired length starting from constructs containing short repeat tracts. We demonstrated that the SLIP method is a time- and cost-effective approach to manipulate the lengths of expanded repeat sequences.

MET-activating Residues in the B-repeat of the Listeria monocytogenes Invasion Protein InlB*

PubMed Central

Bleymüller, Willem M.; Lämmermann, Nina; Ebbes, Maria; Maynard, Daniel; Geerds, Christina; Niemann, Hartmut H.

2016-01-01

The facultative intracellular pathogen Listeria monocytogenes causes listeriosis, a rare but life-threatening disease. Host cell entry begins with activation of the human receptor tyrosine kinase MET through the bacterial invasion protein InlB, which contains an internalin domain, a B-repeat, and three GW domains. The internalin domain is known to bind MET, but no interaction partner is known for the B-repeat. Adding the B-repeat to the internalin domain potentiates MET activation and is required to stimulate Madin-Darby canine kidney (MDCK) cell scatter. Therefore, it has been hypothesized that the B-repeat may bind a co-receptor on host cells. To test this hypothesis, we mutated residues that might be important for binding an interaction partner. We identified two adjacent residues in strand β2 of the β-grasp fold whose mutation abrogated induction of MDCK cell scatter. Biophysical analysis indicated that these mutations do not alter protein structure. We then tested these mutants in human HT-29 cells that, in contrast to the MDCK cells, were responsive to the internalin domain alone. These assays revealed a dominant negative effect, reducing the activity of a construct of the internalin domain and mutated B-repeat below that of the individual internalin domain. Phosphorylation assays of MET and its downstream targets AKT and ERK confirmed the dominant negative effect. Attempts to identify a host cell receptor for the B-repeat were not successful. We conclude that there is limited support for a co-receptor hypothesis and instead suggest that the B-repeat contributes to MET activation through low affinity homodimerization. PMID:27789707

Small glutamine-rich tetratricopeptide repeat-containing protein alpha is present in human ovaries but may not be differentially expressed in relation to polycystic ovary syndrome.

PubMed

Butler, Miriam S; Yang, Xing; Ricciardelli, Carmela; Liang, Xiaoyan; Norman, Robert J; Tilley, Wayne D; Hickey, Theresa E

2013-06-01

To evaluate the expression and function of small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA), an androgen receptor (AR) molecular chaperone, in human ovarian tissues. Examine the effect of SGTA on AR subcellular localization in granulosa tumor cells (KGN) and SGTA expression in ovarian tissues. University-based research laboratory. Archived tissues from premenopausal women and granulosa cells from infertile women receiving assisted reproduction. None. AR subcellular localization and SGTA protein or mRNA levels. SGTA and AR proteins were expressed in the cytoplasm of KGN cells and exposure to androgen stimulated AR nuclear localization. SGTA protein knockdown increased AR nuclear localization at low (0-0.1 nmol/L) but not high (1-10 nmol/L) concentrations of androgen hormone. In ovarian tissues, SGTA was localized to the cytoplasm of granulosa cells at all stages of folliculogenesis and in thecal cells of antral follicles. SGTA protein levels were similar when comparing primordial and primary follicles within core biopsies (n = 40) from women with and without polycystic ovary syndrome (PCOS). Likewise, SGTA mRNA levels were not significantly different in granulosa cells from preovulatory follicles after hyperstimulation of women with and without PCOS. SGTA is present in human ovaries and has the potential to modulate AR signalling, but it may not be differentially expressed in PCOS. Copyright © 2013 American Society for Reproductive Medicine. All rights reserved.

aPPRove: An HMM-Based Method for Accurate Prediction of RNA-Pentatricopeptide Repeat Protein Binding Events

PubMed Central

Harrison, Thomas; Ruiz, Jaime; Sloan, Daniel B.; Ben-Hur, Asa; Boucher, Christina

2016-01-01

Pentatricopeptide repeat containing proteins (PPRs) bind to RNA transcripts originating from mitochondria and plastids. There are two classes of PPR proteins. The P class contains tandem P-type motif sequences, and the PLS class contains alternating P, L and S type sequences. In this paper, we describe a novel tool that predicts PPR-RNA interaction; specifically, our method, which we call aPPRove, determines where and how a PLS-class PPR protein will bind to RNA when given a PPR and one or more RNA transcripts by using a combinatorial binding code for site specificity proposed by Barkan et al. Our results demonstrate that aPPRove successfully locates how and where a PPR protein belonging to the PLS class can bind to RNA. For each binding event it outputs the binding site, the amino-acid-nucleotide interaction, and its statistical significance. Furthermore, we show that our method can be used to predict binding events for PLS-class proteins using a known edit site and the statistical significance of aligning the PPR protein to that site. In particular, we use our method to make a conjecture regarding an interaction between CLB19 and the second intronic region of ycf3. The aPPRove web server can be found at www.cs.colostate.edu/~approve. PMID:27560805

Quantitative interaction screen of telomeric repeat-containing RNA reveals novel TERRA regulators

PubMed Central

Scheibe, Marion; Arnoult, Nausica; Kappei, Dennis; Buchholz, Frank; Decottignies, Anabelle; Butter, Falk; Mann, Matthias

2013-01-01

Telomeres are actively transcribed into telomeric repeat-containing RNA (TERRA), which has been implicated in the regulation of telomere length and heterochromatin formation. Here, we applied quantitative mass spectrometry (MS)–based proteomics to obtain a high-confidence interactome of TERRA. Using SILAC-labeled nuclear cell lysates in an RNA pull-down experiment and two different salt conditions, we distinguished 115 proteins binding specifically to TERRA out of a large set of background binders. While TERRA binders identified in two previous studies showed little overlap, using quantitative mass spectrometry we obtained many candidates reported in these two studies. To test whether novel candidates found here are involved in TERRA regulation, we performed an esiRNA-based interference analysis for 15 of them. Knockdown of 10 genes encoding candidate proteins significantly affected total cellular levels of TERRA, and RNAi of five candidates perturbed TERRA recruitment to telomeres. Notably, depletion of SRRT/ARS2, involved in miRNA processing, up-regulated both total and telomere-bound TERRA. Conversely, knockdown of MORF4L2, a component of the NuA4 histone acetyltransferase complex, reduced TERRA levels both globally and for telomere-bound TERRA. We thus identified new proteins involved in the homeostasis and telomeric abundance of TERRA, extending our knowledge of TERRA regulation. PMID:23921659

DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats

PubMed Central

de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

2015-01-01

Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats. PMID:26481363

Low temperature growth of Ga 1- xIn xP bulk crystals from InSb-rich melt

NASA Astrophysics Data System (ADS)

Gennett, A.; Lewis, D.; Dutta, P. S.

2010-04-01

Bulk growth of phosphorus and arsenic based ternary III-V semiconductor crystals using pseudo-binary melts such as GaP-InP, GaP-GaAs, AlAs-GaAs, etc. is significantly challenging due to the high vapor pressures of group V species in conjunction with slow growth rates and the need for melt replenishment and mixing during growth. Lowering the growth temperature is desirable such that the vapor pressures of P and As can be easily handled. Low growth temperatures could be achieved by using Ga or In rich solutions. However, this approach is less attractive for growing bulk crystals due to several experimental difficulties including sticking of the growth solution to the crucible wall and to the grown crystal, making it challenging for crystal extraction. Growth of ternary crystals from low temperature quaternary melts has been found to be attractive. In this paper, we will present a new method for the growth of Ga 1- xIn xP from InSb rich Ga 1- xIn xP ySb 1- y melts at low growth temperatures in the range of 800-1050 °C. Thermodynamic phase diagrams calculated at various temperatures using a Gibbs free energy minimization software and materials databases commercially available from Thermo-Calc software will be presented along with experimental validation for Ga 1- xIn xP crystals grown at 1000 °C.

DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats.

PubMed

de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

2015-11-16

Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Insights into the Aggregation Mechanism of PolyQ Proteins with Different Glutamine Repeat Lengths.

PubMed

Yushchenko, Tetyana; Deuerling, Elke; Hauser, Karin

2018-04-24

Polyglutamine (polyQ) diseases, including Huntington's disease, result from the aggregation of an abnormally expanded polyQ repeat in the affected protein. The length of the polyQ repeat is essential for the disease's onset; however, the molecular mechanism of polyQ aggregation is still poorly understood. Controlled conditions and initiation of the aggregation process are prerequisites for the detection of transient intermediate states. We present an attenuated total reflection Fourier-transform infrared spectroscopic approach combined with protein immobilization to study polyQ aggregation dependent on the polyQ length. PolyQ proteins were engineered mimicking the mammalian N-terminus fragment of the Huntingtin protein and containing a polyQ sequence with the number of glutamines below (Q11), close to (Q38), and above (Q56) the disease threshold. A monolayer of the polyQ construct was chemically immobilized on the internal reflection element of the attenuated total reflection cell, and the aggregation was initiated via enzymatic cleavage. Structural changes of the polyQ sequence were monitored by time-resolved infrared difference spectroscopy. We observed faster aggregation kinetics for the longer sequences, and furthermore, we could distinguish β-structured intermediates for the different constructs, allowing us to propose aggregation mechanisms dependent on the repeat length. Q11 forms a β-structured aggregate by intermolecular interaction of stretched monomers, whereas Q38 and Q56 undergo conformational changes to various β-structured intermediates, including intramolecular β-sheets. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Sel1-like repeat proteins in signal transduction.

PubMed

Mittl, Peer R E; Schneider-Brachert, Wulf

2007-01-01

Solenoid proteins, which are distinguished from general globular proteins by their modular architectures, are frequently involved in signal transduction pathways. Proteins from the tetratricopeptide repeat (TPR) and Sel1-like repeat (SLR) families share similar alpha-helical conformations but different consensus sequence lengths and superhelical topologies. Both families are characterized by low sequence similarity levels, rendering the identification of functional homologous difficult. Therefore current knowledge of the molecular and cellular functions of the SLR proteins Sel1, Hrd3, Chs4, Nif1, PodJ, ExoR, AlgK, HcpA, Hsp12, EnhC, LpnE, MotX, and MerG has been reviewed. Although SLR proteins possess different cellular functions they all seem to serve as adaptor proteins for the assembly of macromolecular complexes. Sel1, Hrd3, Hsp12 and LpnE are activated under cellular stress. The eukaryotic Sel1 and Hrd3 proteins are involved in the ER-associated protein degradation, whereas the bacterial LpnE, EnhC, HcpA, ExoR, and AlgK proteins mediate the interactions between bacterial and eukaryotic host cells. LpnE and EnhC are responsible for the entry of L. pneumophila into epithelial cells and macrophages. ExoR from the symbiotic microorganism S. melioti and AlgK from the pathogen P. aeruginosa regulate exopolysaccaride synthesis. Nif1 and Chs4 from yeast are responsible for the regulation of mitosis and septum formation during cell division, respectively, and PodJ guides the cellular differentiation during the cell cycle of the bacterium C. crescentus. Taken together the SLR motif establishes a link between signal transduction pathways from eukaryotes and bacteria. The SLR motif is so far absent from archaea. Therefore the SLR could have developed in the last common ancestor between eukaryotes and bacteria.

Macrocycles that inhibit the binding between heat shock protein 90 and TPR-containing proteins

PubMed Central

Ardi, Veronica C.; Alexander, Leslie D.; Johnson, Victoria; McAlpine, Shelli R.

2011-01-01

Heat shock protein 90 (Hsp90) accounts for 1–2% of the total proteins in normal cells and functions as a molecular chaperone that folds, assembles, and stabilizes client proteins. Hsp90 is over-expressed (3–6-fold increase) in stressed cells, including cancer cells, and regulates over 200 client and co-chaperone proteins. Hsp90 client proteins are involved in a plethora of cellular signaling events including numerous growth and apoptotic pathways. Since pathway-specific inhibitors can be problematic in drug-resistant cancers, shutting down multiple pathways at once is a promising approach when developing new therapeutics. Hsp90’s ability to modulate many growth and signaling pathways simultaneously makes this protein an attractive target in the field of cancer therapeutics. Herein we present evidence that a small molecule modulates Hsp90 via binding between the N and middle domain and allosterically inhibiting the binding interaction between Hsp90 and four C-terminal binding client proteins: IP6K2, FKBP38, FKBP52, and HOP. These last three clients contain a tetratricopeptide-repeat (TPR) region, which is known to interact with the MEEVD sequence on the C-terminus of Hsp90. Thus, this small molecule modulates the activity between co-chaperones that contain TPR motifs and Hsp90’s MEEVD region. This mechanism of action is unique from that of all Hsp90 inhibitors currently in clinical trials where these molecules have no effect on proteins that bind to the C-terminus of Hsp90. Further, our small molecule induces a Caspase-3 dependent apoptotic event. Thus, we describe the mechanism of a novel scaffold that is a useful tool for studying cell-signaling events that result when blocking the MEEVD-TPR interaction between Hsp90 and co-chaperone proteins. PMID:21950602

MSH3 polymorphisms and protein levels affect CAG repeat instability in Huntington's disease mice.

PubMed

Tomé, Stéphanie; Manley, Kevin; Simard, Jodie P; Clark, Greg W; Slean, Meghan M; Swami, Meera; Shelbourne, Peggy F; Tillier, Elisabeth R M; Monckton, Darren G; Messer, Anne; Pearson, Christopher E

2013-01-01

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of

MSH3 Polymorphisms and Protein Levels Affect CAG Repeat Instability in Huntington's Disease Mice

PubMed Central

Simard, Jodie P.; Clark, Greg W.; Slean, Meghan M.; Swami, Meera; Shelbourne, Peggy F.; Tillier, Elisabeth R. M.; Monckton, Darren G.; Messer, Anne; Pearson, Christopher E.

2013-01-01

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of

A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization

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

Qiu, Chen; McCann, Kathleen L.; Wine, Robert N.

Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins bind sequence specifically to mRNA targets using a single-stranded RNA-binding domain comprising eight Pumilio (PUM) repeats. PUM repeats have now been identified in proteins that function in pre-rRNA processing, including human Puf-A and yeast Puf6. This is a role not previously ascribed to PUF proteins. In this paper we present crystal structures of human Puf-A that reveal a class of nucleic acid-binding proteins with 11 PUM repeats arranged in an “L”-like shape. In contrast to classical PUF proteins, Puf-A forms sequence-independent interactions with DNA or RNA, mediated by conservedmore » basic residues. We demonstrate that equivalent basic residues in yeast Puf6 are important for RNA binding, pre-rRNA processing, and mRNA localization. Finally, PUM repeats can be assembled into alternative folds that bind to structured nucleic acids in addition to forming canonical eight-repeat crescent-shaped RNA-binding domains found in classical PUF proteins.« less

A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization

DOE PAGES

Qiu, Chen; McCann, Kathleen L.; Wine, Robert N.; ...

2014-12-15

Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins bind sequence specifically to mRNA targets using a single-stranded RNA-binding domain comprising eight Pumilio (PUM) repeats. PUM repeats have now been identified in proteins that function in pre-rRNA processing, including human Puf-A and yeast Puf6. This is a role not previously ascribed to PUF proteins. In this paper we present crystal structures of human Puf-A that reveal a class of nucleic acid-binding proteins with 11 PUM repeats arranged in an “L”-like shape. In contrast to classical PUF proteins, Puf-A forms sequence-independent interactions with DNA or RNA, mediated by conservedmore » basic residues. We demonstrate that equivalent basic residues in yeast Puf6 are important for RNA binding, pre-rRNA processing, and mRNA localization. Finally, PUM repeats can be assembled into alternative folds that bind to structured nucleic acids in addition to forming canonical eight-repeat crescent-shaped RNA-binding domains found in classical PUF proteins.« less

Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins

PubMed Central

Jung, Huihun; Pena-Francesch, Abdon; Saadat, Alham; Sebastian, Aswathy; Kim, Dong Hwan; Hamilton, Reginald F.; Albert, Istvan; Allen, Benjamin D.; Demirel, Melik C.

2016-01-01

Many globular and structural proteins have repetitions in their sequences or structures. However, a clear relationship between these repeats and their contribution to the mechanical properties remains elusive. We propose a new approach for the design and production of synthetic polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions. Our designed sequences are based on a structural protein produced in squid suction cups that has a segmented copolymer structure with amorphous and crystalline domains. We produced segmented polypeptides with varying repeat number, while keeping the lengths and compositions of the amorphous and crystalline regions fixed. We showed that mechanical properties of these synthetic proteins could be tuned by modulating their molecular weights. Specifically, the toughness and extensibility of synthetic polypeptides increase as a function of the number of tandem repeats. This result suggests that the repetitions in native squid proteins could have a genetic advantage for increased toughness and flexibility. PMID:27222581

Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat-expanded C9orf72 amyotrophic lateral sclerosis.

PubMed

Saberi, Shahram; Stauffer, Jennifer E; Jiang, Jie; Garcia, Sandra Diaz; Taylor, Amy E; Schulte, Derek; Ohkubo, Takuya; Schloffman, Cheyenne L; Maldonado, Marcus; Baughn, Michael; Rodriguez, Maria J; Pizzo, Don; Cleveland, Don; Ravits, John

2018-03-01

Hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (C9 ALS). The main hypothesized pathogenic mechanisms are C9orf72 haploinsufficiency and/or toxicity from one or more of bi-directionally transcribed repeat RNAs and their dipeptide repeat proteins (DPRs) poly-GP, poly-GA, poly-GR, poly-PR and poly-PA. Recently, nuclear import and/or export defects especially caused by arginine-containing poly-GR or poly-PR have been proposed as significant contributors to pathogenesis based on disease models. We quantitatively studied and compared DPRs, nuclear pore proteins and C9orf72 protein in clinically related and clinically unrelated regions of the central nervous system, and compared them to phosphorylated TDP-43 (pTDP-43), the hallmark protein of ALS. Of the five DPRs, only poly-GR was significantly abundant in clinically related areas compared to unrelated areas (p < 0.001), and formed dendritic-like aggregates in the motor cortex that co-localized with pTDP-43 (p < 0.0001). While most poly-GR dendritic inclusions were pTDP-43 positive, only 4% of pTDP-43 dendritic inclusions were poly-GR positive. Staining for arginine-containing poly-GR and poly-PR in nuclei of neurons produced signals that were not specific to C9 ALS. We could not detect significant differences of nuclear markers RanGap, Lamin B1, and Importin β1 in C9 ALS, although we observed subtle nuclear changes in ALS, both C9 and non-C9, compared to control. The C9orf72 protein itself was diffusely expressed in cytoplasm of large neurons and glia, and nearly 50% reduced, in both clinically related frontal cortex and unrelated occipital cortex, but not in cerebellum. In summary, sense-encoded poly-GR DPR was unique, and localized to dendrites and pTDP43 in motor regions of C9 ALS CNS. This is consistent with new emerging ideas about TDP-43 functions in dendrites.

Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat expanded C9orf72 amyotrophic lateral sclerosis

PubMed Central

Saberi, Shahram; Stauffer, Jennifer E.; Jiang, Jie; Garcia, Sandra Diaz; Taylor, Amy E; Schulte, Derek; Ohkubo, Takuya; Schloffman, Cheyenne L.; Maldonado, Marcus; Baughn, Michael; Rodriguez, Maria J; Pizzo, Don; Cleveland, Don; Ravits, John

2018-01-01

Hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (C9 ALS). The main hypothesized pathogenic mechanisms are C9orf72 haploinsufficiency and/or toxicity from one or more of bi-directionally transcribed repeat RNAs and their dipeptide repeat proteins (DPRs) poly-GP, poly-GA, poly-GR, poly-PR and poly-PA. Recently, nuclear import and/or export defects especially caused by arginine-containing poly-GR or poly-PR have been proposed as significant contributors to pathogenesis based on disease models. We quantitatively studied and compared DPRs, nuclear pore proteins and C9orf72 protein in clinically-related and clinically-unrelated regions of the central nervous system, and compared them to phosphorylated TDP-43 (pTDP-43), the hallmark protein of ALS. Of the five DPRs, only poly-GR was significantly abundant in clinically-related areas compared to unrelated areas (p<0.001), and formed dendritic-like aggregates in the motor cortex that co-localized with pTDP-43 (p<0.0001). While most poly-GR dendritic inclusions were pTDP-43-positive, only 4% of pTDP-43 dendritic inclusions were poly-GR-positive. Staining for arginine-containing poly-GR and poly-PR in nuclei of neurons produced signals that were not specific to C9 ALS. We could not detect significant differences of nuclear markers RanGap, Lamin B1, and Importin β1 in C9 ALS, although we observed subtle nuclear changes in ALS, both C9 and non-C9, compared to control. The C9orf72 protein itself was diffusely expressed in cytoplasm of large neurons and glia, and nearly 50% reduced, in both clinically-related frontal cortex and unrelated occipital cortex, but not in cerebellum. In summary, sense-encoded poly-GR DPR was unique, and localized to neurites and pTDP43 in motor regions of C9 ALS CNS. This is consistent with new emerging ideas about TDP-43 functions in dendrites. PMID:29196813

Leucine-rich repeat-containing G protein-coupled receptor 4 facilitates vesicular stomatitis virus infection by binding vesicular stomatitis virus glycoprotein.

PubMed

Zhang, Na; Huang, Hongjun; Tan, Binghe; Wei, Yinglei; Xiong, Qingqing; Yan, Yan; Hou, Lili; Wu, Nannan; Siwko, Stefan; Cimarelli, Andrea; Xu, Jianrong; Han, Honghui; Qian, Min; Liu, Mingyao; Du, Bing

2017-10-06

Vesicular stomatitis virus (VSV) and rabies and Chandipura viruses belong to the Rhabdovirus family. VSV is a common laboratory virus to study viral evolution and host immune responses to viral infection, and recombinant VSV-based vectors have been widely used for viral oncolysis, vaccination, and gene therapy. Although the tropism of VSV is broad, and its envelope glycoprotein G is often used for pseudotyping other viruses, the host cellular components involved in VSV infection remain unclear. Here, we demonstrate that the host protein leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is essential for VSV and VSV-G pseudotyped lentivirus (VSVG-LV) to infect susceptible cells. Accordingly, Lgr4-deficient mice had dramatically decreased VSV levels in the olfactory bulb. Furthermore, Lgr4 knockdown in RAW 264.7 cells also significantly suppressed VSV infection, and Lgr4 overexpression in RAW 264.7 cells enhanced VSV infection. Interestingly, only VSV infection relied on Lgr4, whereas infections with Newcastle disease virus, influenza A virus (A/WSN/33), and herpes simplex virus were unaffected by Lgr4 status. Of note, assays of virus entry, cell ELISA, immunoprecipitation, and surface plasmon resonance indicated that VSV bound susceptible cells via the Lgr4 extracellular domain. Pretreating cells with an Lgr4 antibody, soluble LGR4 extracellular domain, or R-spondin 1 blocked VSV infection by competitively inhibiting VSV binding to Lgr4. Taken together, the identification of Lgr4 as a VSV-specific host factor provides important insights into understanding VSV entry and its pathogenesis and lays the foundation for VSV-based gene therapy and viral oncolytic therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of muscle ankyrin repeat proteins in human skeletal muscle.

PubMed

Wette, Stefan G; Smith, Heather K; Lamb, Graham D; Murphy, Robyn M

2017-09-01

Muscle ankyrin repeat proteins (MARPs) are a family of titin-associated, stress-response molecules and putative transducers of stretch-induced signaling in skeletal muscle. In cardiac muscle, cardiac ankyrin repeat protein (CARP) and diabetes-related ankyrin repeat protein (DARP) reportedly redistribute from binding sites on titin to the nucleus following a prolonged stretch. However, it is unclear whether ankyrin repeat domain protein 2 (Ankrd 2) shows comparable stretch-induced redistribution to the nucleus. We measured the following in rested human skeletal muscle: 1 ) the absolute amount of MARPs and 2 ) the distribution of Ankrd 2 and DARP in both single fibers and whole muscle preparations. In absolute amounts, Ankrd 2 is the most abundant MARP in human skeletal muscle, there being ~3.1 µmol/kg, much greater than DARP and CARP (~0.11 and ~0.02 µmol/kg, respectively). All DARP was found to be tightly bound at cytoskeletal (or possibly nuclear) sites. In contrast, ~70% of the total Ankrd 2 is freely diffusible in the cytosol [including virtually all of the phosphorylated (p)Ankrd 2-Ser99 form], ~15% is bound to non-nuclear membranes, and ~15% is bound at cytoskeletal sites, likely at the N2A region of titin. These data are not consistent with the proposal that Ankrd 2, per se, or pAnkrd 2-Ser99 mediates stretch-induced signaling in skeletal muscle, dissociating from titin and translocating to the nucleus, because the majority of these forms of Ankrd 2 are already free in the cytosol. It will be necessary to show that the titin-associated Ankrd 2 is modified by stretch in some as-yet-unidentified way, distinct from the diffusible pool, if it is to act as a stretch-sensitive signaling molecule. Copyright © 2017 the American Physiological Society.

Proteins containing expanded polyglutamine tracts and neurodegenerative disease

PubMed Central

Adegbuyiro, Adewale; Sedighi, Faezeh; Pilkington, Albert W.; Groover, Sharon; Legleiter, Justin

2017-01-01

Several hereditary neurological and neuromuscular diseases are caused by an abnormal expansion of trinucleotide repeats. To date, there have been ten of these trinucleotide repeat disorders associated with an expansion of the codon CAG encoding glutamine (Q). For these polyglutamine (polyQ) diseases, there is a critical threshold length of the CAG repeat required for disease, and further expansion beyond this threshold is correlated with age of onset and symptom severity. PolyQ expansion in the translated proteins promotes their self-assembly into a variety of oligomeric and fibrillar aggregate species that accumulate into the hallmark proteinaceous inclusion bodies associated with each disease. Here, we review aggregation mechanisms of proteins with expanded polyQ-tracts, structural consequences of expanded polyQ ranging from monomers to fibrillar aggregates, the impact of protein context and post translational modifications on aggregation, and a potential role for lipids membranes in aggregation. As the pathogenic mechanisms that underlie these disorders are often classified as either a gain of toxic function or loss of normal protein function, some toxic mechanisms associated with mutant polyQ tracts will also be discussed. PMID:28170216

[Convergent origin of repeats in genes coding for globular proteins. An analysis of the factors determining the presence of inverted and symmetrical repeats].

PubMed

Solov'ev, V V; Kel', A E; Kolchanov, N A

1989-01-01

The factors, determining the presence of inverted and symmetrical repeats in genes coding for globular proteins, have been analysed. An interesting property of genetical code has been revealed in the analysis of symmetrical repeats: the pairs of symmetrical codons corresponded to pairs of amino acids with mostly similar physical-chemical parameters. This property may explain the presence of symmetrical repeats and palindromes only in genes coding for beta-structural proteins-polypeptides, where amino acids with similar physical-chemical properties occupy symmetrical positions. A stochastic model of evolution of polynucleotide sequences has been used for analysis of inverted repeats. The modelling demonstrated that only limiting of sequences (uneven frequencies of used codons) is enough for arising of nonrandom inverted repeats in genes.

The Mr 140,000 Intermediate Chain of Chlamydomonas Flagellar Inner Arm Dynein Is a WD-Repeat Protein Implicated in Dynein Arm Anchoring

PubMed Central

Yang, Pinfen; Sale, Winfield S.

1998-01-01

Previous structural and biochemical studies have revealed that the inner arm dynein I1 is targeted and anchored to a unique site located proximal to the first radial spoke in each 96-nm axoneme repeat on flagellar doublet microtubules. To determine whether intermediate chains mediate the positioning and docking of dynein complexes, we cloned and characterized the 140-kDa intermediate chain (IC140) of the I1 complex. Sequence and secondary structural analysis, with particular emphasis on β-sheet organization, predicted that IC140 contains seven WD repeats. Reexamination of other members of the dynein intermediate chain family of WD proteins indicated that these polypeptides also bear seven WD/β-sheet repeats arranged in the same pattern along each intermediate chain protein. A polyclonal antibody was raised against a 53-kDa fusion protein derived from the C-terminal third of IC140. The antibody is highly specific for IC140 and does not bind to other dynein intermediate chains or proteins in Chlamydomonas flagella. Immunofluorescent microscopy of Chlamydomonas cells confirmed that IC140 is distributed along the length of both flagellar axonemes. In vitro reconstitution experiments demonstrated that the 53-kDa C-terminal fusion protein binds specifically to axonemes lacking the I1 complex. Chemical cross-linking indicated that IC140 is closely associated with a second intermediate chain in the I1 complex. These data suggest that IC140 contains domains responsible for the assembly and docking of the I1 complex to the doublet microtubule cargo. PMID:9843573

Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth.

PubMed

Draeger, Christian; Ndinyanka Fabrice, Tohnyui; Gineau, Emilie; Mouille, Grégory; Kuhn, Benjamin M; Moller, Isabel; Abdou, Marie-Therese; Frey, Beat; Pauly, Markus; Bacic, Antony; Ringli, Christoph

2015-06-24

Leucine-rich repeat extensins (LRXs) are extracellular proteins consisting of an N-terminal leucine-rich repeat (LRR) domain and a C-terminal extensin domain containing the typical features of this class of structural hydroxyproline-rich glycoproteins (HRGPs). The LRR domain is likely to bind an interaction partner, whereas the extensin domain has an anchoring function to insolubilize the protein in the cell wall. Based on the analysis of the root hair-expressed LRX1 and LRX2 of Arabidopsis thaliana, LRX proteins are important for cell wall development. The importance of LRX proteins in non-root hair cells and on the structural changes induced by mutations in LRX genes remains elusive. The LRX gene family of Arabidopsis consists of eleven members, of which LRX3, LRX4, and LRX5 are expressed in aerial organs, such as leaves and stem. The importance of these LRX genes for plant development and particularly cell wall formation was investigated. Synergistic effects of mutations with gradually more severe growth retardation phenotypes in double and triple mutants suggest a similar function of the three genes. Analysis of cell wall composition revealed a number of changes to cell wall polysaccharides in the mutants. LRX3, LRX4, and LRX5, and most likely LRX proteins in general, are important for cell wall development. Due to the complexity of changes in cell wall structures in the lrx mutants, the exact function of LRX proteins remains to be determined. The increasingly strong growth-defect phenotypes in double and triple mutants suggests that the LRX proteins have similar functions and that they are important for proper plant development.

Influence of repeated infusion of capsaicin-contained red pepper sauce on esophageal secondary peristalsis in humans.

PubMed

Liu, T T; Yi, C H; Lei, W Y; Hung, X S; Yu, H C; Chen, C L

2014-10-01

The transient receptor potential vanilloid 1 has been implicated as a target mediator for heartburn perception and modulation of esophageal secondary peristalsis. Our aim was to determine the effect of repeated esophageal infusion of capsaicin-contained red pepper sauce on heartburn perception and secondary peristalsis in healthy adults. Secondary peristalsis was performed with mid-esophageal injections of air in 15 healthy adults. Two separate protocols including esophageal infusion with saline and capsaicin-contained red pepper sauce and 2 consecutive sessions of capsaicin-contained red pepper sauce were randomly performed. After repeated infusion of capsaicin-contained red pepper sauce, the threshold volume to activate secondary peristalsis was significantly increased during slow (p < 0.001) and rapid air injections (p = 0.004). Acute infusion of capsaicin-contained red pepper sauce enhanced heartburn perception (p < 0.001), but the intensity of heartburn perception was significantly reduced after repeated capsaicin-contained red pepper sauce infusion (p = 0.007). Acute infusion of capsaicin-contained red pepper sauce significantly increased pressure wave amplitudes of distal esophagus during slow (p = 0.003) and rapid air injections (p = 0.01), but repeated infusion of capsaicin-contained red pepper sauce significantly decreased pressure wave amplitude of distal esophagus during slow (p = 0.0005) and rapid air injections (p = 0.003). Repeated esophageal infusion of capsaicin appears to attenuate heartburn perception and inhibit distension-induced secondary peristalsis in healthy adults. These results suggest capsaicin-sensitive afferents in modulating sensorimotor function of secondary peristalsis in human esophagus. © 2014 John Wiley & Sons Ltd.

A Legionella pneumophila collagen-like protein encoded by a gene with a variable number of tandem repeats is involved in the adherence and invasion of host cells.

PubMed

Vandersmissen, Liesbeth; De Buck, Emmy; Saels, Veerle; Coil, David A; Anné, Jozef

2010-05-01

Legionella pneumophila is a Gram-negative, facultative intracellular pathogen and the causative agent of Legionnaires' disease, a severe pneumonia in humans. Analysis of the Legionella sequenced genomes revealed a gene with a variable number of tandem repeats (VNTRs), whose number varies between strains. We examined the strain distribution of this gene among a collection of 108 clinical, environmental and hot spring serotype I strains. Twelve variants were identified, but no correlation was observed between the number of repeat units and clinical and environmental strains. The encoded protein contains the C-terminal consensus motif of outer membrane proteins and has a large region of collagen-like repeats that is encoded by the VNTR region. We have therefore annotated this protein Lcl for Legionella collagen-like protein. Lcl was shown to contribute to the adherence and invasion of host cells and it was demonstrated that the number of repeat units present in lcl had an influence on these adhesion characteristics.

Field performance of Quercus bicolor established as repeatedly air-root-pruned container and bareroot planting stock

Treesearch

J.W." Jerry" Van Sambeek; Larry D. Godsey; William D. Walter; Harold E. Garrett; John P. Dwyer

2016-01-01

Benefits of repeated air-root-pruning of seedlings when stepping up to progressively larger containers include excellent lateral root distribution immediately below the root collar and an exceptionally fibrous root ball. To evaluate long-term field performance of repeatedly air-root-pruned container stock, three plantings of swamp white oak (Quercus bicolor...

Blends of cysteine-containing proteins

NASA Astrophysics Data System (ADS)

Barone, Justin

2005-03-01

Many agricultural wastes are made of proteins such as keratin, lactalbumin, gluten, and albumin. These proteins contain the amino acid cysteine. Cysteine allows for the formation of inter-and intra-molecular sulfur-sulfur bonds. Correlations are made between the properties of films made from the proteins and the amino acid sequence. Blends of cysteine-containing proteins show possible synergies in physical properties at intermediate concentrations. FT-IR spectroscopy shows increased hydrogen bonding at intermediate concentrations suggesting that this contributes to increased physical properties. DSC shows limited miscibility and the formation of new crystalline phases in the blends suggesting that this too contributes.

Core Histones and HIRIP3, a Novel Histone-Binding Protein, Directly Interact with WD Repeat Protein HIRA

PubMed Central

Lorain, Stéphanie; Quivy, Jean-Pierre; Monier-Gavelle, Frédérique; Scamps, Christine; Lécluse, Yann; Almouzni, Geneviève; Lipinski, Marc

1998-01-01

The human HIRA gene has been named after Hir1p and Hir2p, two corepressors which together appear to act on chromatin structure to control gene transcription in Saccharomyces cerevisiae. HIRA homologs are expressed in a regulated fashion during mouse and chicken embryogenesis, and the human gene is a major candidate for the DiGeorge syndrome and related developmental disorders caused by a reduction to single dose of a fragment of chromosome 22q. Western blot analysis and double-immunofluorescence experiments using a specific antiserum revealed a primary nuclear localization of HIRA. Similar to Hir1p, HIRA contains seven amino-terminal WD repeats and probably functions as part of a multiprotein complex. HIRA and core histone H2B were found to physically interact in a yeast double-hybrid protein interaction trap, in GST pull-down assays, and in coimmunoprecipitation experiments performed from cellular extracts. In vitro, HIRA also interacted with core histone H4. H2B- and H4-binding domains were overlapping but distinguishable in the carboxy-terminal region of HIRA, and the region for HIRA interaction was mapped to the amino-terminal tail of H2B and the second α helix of H4. HIRIP3 (HIRA-interacting protein 3) is a novel gene product that was identified from its HIRA-binding properties in the yeast protein interaction trap. In vitro, HIRIP3 directly interacted with HIRA but also with core histones H2B and H3, suggesting that a HIRA-HIRIP3-containing complex could function in some aspects of chromatin and histone metabolism. Insufficient production of HIRA, which we report elsewhere interacts with homeodomain-containing DNA-binding factors during mammalian embryogenesis, could perturb the stoichiometric assembly of multimolecular complexes required for normal embryonic development. PMID:9710638

Nucleotide sequences of Dictyostelium discoideum developmentally regulated cDNAs rich in (AAC) imply proteins that contain clusters of asparagine, glutamine, or threonine.

PubMed

Shaw, D R; Richter, H; Giorda, R; Ohmachi, T; Ennis, H L

1989-09-01

A Dictyostelium discoideum repetitive element composed of long repeats of the codon (AAC) is found in developmentally regulated transcripts. The concentration of (AAC) sequences is low in mRNA from dormant spores and growing cells and increases markedly during spore germination and multicellular development. The sequence hybridizes to many different sized Dictyostelium DNA restriction fragments indicating that it is scattered throughout the genome. Four cDNA clones isolated contain (AAC) sequences in the deduced coding region. Interestingly, the (AAC)-rich sequences are present in all three reading frames in the deduced proteins, i.e., AAC (asparagine), ACA (threonine) and CAA (glutamine). Three of the clones contain only one of these in-frame so that the individual proteins carry either asparagine, threonine, or glutamine clusters, not mixtures. However, one clone is both glutamine- and asparagine-rich. The (AAC) portion of the transcripts are reiterated 300 times in the haploid genome while the other portions of the cDNAs represent single copy genes, whose sequences show no similarity other than the (AAC) repeats. The repeated sequence is similar to the opa or M sequence found in Drosophila melanogaster notch and homeo box genes and in fly developmentally regulated transcripts. The transcripts are present on polysomes suggesting that they are translated. Although the function of these repeats is unknown, long amino acid repeats are a characteristic feature of extracellular proteins of lower eukaryotes.

[An electron microscopy study of the structure of polyelectrolyte microcapsules containing protein and containing no protein].

PubMed

Kazakova, L I; Dubrovskiĭ, A V; Moshkov, D A; Shabarchina, L I; Sukhorukov, B I

2007-01-01

Electron micrographs of ultrathin sections of polyelectrolyte microparticles containing protein and free from protein for the formation of which CaCO3 spherulites served as a core basis have been obtained and analyzed. Polyelectrolyte microparticles with the number of alternately layered polyelectrolyte layers of polystyrene sulfonate and polyallylamine from 6 to 11 have been studied. It follows from the data obtained that protein-free polyelectrolyte particles having the dimensions 4.5-5 mm are formations of an intricate internal organization, which consist of a set of threadlike and closed nanoelements of polyelectrolyte nature with a thickness of 20-30 nm. The particles containing six to eight polyelectrolyte layers lack the external envelope; therefore, they were called polyelectrolyte microspherulites. With the number of layers nine and more, when a polyelectrolyte envelope appears on the surface, they transfer into polyelectrolyte microcapsules. It was found that, in a protein-containing polyelectrolyte microcapsule, as distinct from protein-free polyelectrolyte microspherulite and microcapsule, polyelectrolytes are located only in the nearsurface layer, and the external spatially organized envelope restricts the internal volume filled with protein solution. As the number of polyelectrolyte layers increases, the thickness of the envelope increases. The reasons for such substantial differences in the structures of polyelectrolyte microcapsules formed on protein-containing and protein-free CaCO3 spherulite are discussed.

SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis.

PubMed Central

Jacobsen, S E; Binkowski, K A; Olszewski, N E

1996-01-01

Gibberellins (GAs) are a major class of plant hormones that control many developmental processes, including seed development and germination, flower and fruit development, and flowering time. Genetic studies with Arabidopsis thaliana have identified two genes involved in GA perception or signal transduction. A semidominant mutation at the GIBBERELLIN INSENSITIVE (GAI) locus results in plants resembling GA-deficient mutants but exhibiting reduced sensitivity to GA. Recessive mutations at the SPINDLY (SPY) locus cause a phenotype that is consistent with constitutive activation of GA signal transduction. Here we show that a strong allele of spy is completely epistatic to gai, indicating that SPY acts downstream of GAI. We have cloned the SPY gene and shown that it encodes a new type of signal transduction protein, which contains a tetratricopeptide repeat region, likely serving as a protein interaction domain, and a novel C-terminal region. Mutations in both domains increase GA signal transduction. The presence of a similar gene in Caenorhabditis elegans suggests that SPY represents a class of signal transduction proteins that is present throughout the eukaryotes. Images Fig. 1 Fig. 2 Fig. 3 PMID:8799194

Study of vibrational modes in CuxAg1-xIn5S8 mixed crystals by infrared reflection measurements

NASA Astrophysics Data System (ADS)

Gasanly, N. M.

2018-04-01

Infrared reflection spectra of CuxAg1-xIn5S8 mixed crystals, grown by Bridgman method, were studied in the wide frequency range of 50-2000 cm-1. All four infrared-active modes were detected, which are in full agreement with the prediction of group-theoretical analysis. Real and imaginary parts of the dielectric function, refractive index and the energy losses function were evaluated from reflectivity measurements. The frequencies of TO and LO modes and oscillator strengths were also determined. The bands detected in IR spectra of studied crystals were assigned to various vibration types (valence and valence-deformation) on the basis of the symmetrized displacements of atoms obtained employing the Melvin projection operators. The linear dependencies of optical mode frequencies on the composition of CuxAg1-xIn5S8 mixed crystals were obtained. These dependencies display one-mode behavior.

The prognostic role of Leucine-rich repeat-containing G-protein-coupled receptor 5 in gastric cancer: A systematic review with meta-analysis.

PubMed

Huang, Tianchen; Qiu, Xinguang; Xiao, Jianan; Wang, Qingbing; Wang, Yanjun; Zhang, Yong; Bai, Dongxiao

2016-04-01

The prognostic value of Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) in gastric cancer remains controversial. To further investigate this relationship, we performed meta-analyses to systematically review the association between LGR5 expression and various clinical parameters in gastric cancer patients. Eligible studies from PubMed, Embase, Web of Science, CNKI (Chinese National Knowledge Infrastructure), Wangfang (Database of Chinese Ministry of Science & Technology) and CBM (China Biological Medicine) databases were evaluated to investigate the association of LGR5 expression with overall survival (OS) and clinicopathological features of gastric cancer. LGR5 overexpression was significantly associated with poor OS in patients with gastric cancer (HR 1.66, 95% CI 1.02-2.69). LGR5 overexpression was also significantly associated with TNM stage (TIII/TIV vs TI/TII: OR 5.42, 95% CI 1.02-28.72) and lymph node metastasis (positive vs negative: OR 2.30, 95% CI 1.06-5.0). Our meta-analysis indicates that LGR5 may be a predictive factor for invasion and metastasis, and poor prognosis in patients with gastric cancer. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

REPETITA: detection and discrimination of the periodicity of protein solenoid repeats by discrete Fourier transform

PubMed Central

Marsella, Luca; Sirocco, Francesco; Trovato, Antonio; Seno, Flavio; Tosatto, Silvio C.E.

2009-01-01

Motivation: Proteins with solenoid repeats evolve more quickly than non-repetitive ones and their periodicity may be rapidly hidden at sequence level, while still evident in structure. In order to identify these repeats, we propose here a novel method based on a metric characterizing amino-acid properties (polarity, secondary structure, molecular volume, codon diversity, electric charge) using five previously derived numerical functions. Results: The five spectra of the candidate sequences coding for structural repeats, obtained by Discrete Fourier Transform (DFT), show common features allowing determination of repeat periodicity with excellent results. Moreover it is possible to introduce a phase space parameterized by two quantities related to the Fourier spectra which allow for a clear distinction between a non-homologous set of globular proteins and proteins with solenoid repeats. The DFT method is shown to be competitive with other state of the art methods in the detection of solenoid structures, while improving its performance especially in the identification of periodicities, since it is able to recognize the actual repeat length in most cases. Moreover it highlights the relevance of local structural propensities in determining solenoid repeats. Availability: A web tool implementing the algorithm presented in the article (REPETITA) is available with additional details on the data sets at the URL: http://protein.bio.unipd.it/repetita/. Contact: silvio.tosatto@unipd.it PMID:19478001

Myotonin protein-kinase [AGC]n trinucleotide repeat in seven nonhuman primates

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

Novelli, G.; Sineo, L.; Pontieri, E.

Myotonic dystrophy (DM) is due to a genomic instability of a trinucleotide [AGC]n motif, located at the 3{prime} UTR region of a protein-kinase gene (myotonin protein kinase, MT-PK). The [AGC] repeat is meiotically and mitotically unstable, and it is directly related to the manifestations of the disorder. Although a gene dosage effect of the MT-PK has been demonstrated n DM muscle, the mechanism(s) by which the intragenic repeat expansion leads to disease is largely unknown. This non-standard mutational event could reflect an evolutionary mechanism widespread among animal genomes. We have isolated and sequenced the complete 3{prime}UTR region of the MT-PKmore » gene in seven primates (macaque, orangutan, gorilla, chimpanzee, gibbon, owl monkey, saimiri), and examined by comparative sequence nucleotide analysis the [AGC]n intragenic repeat and the surrounding nucleotides. The genomic organization, including the [AGC]n repeat structure, was conserved in all examined species, excluding the gibbon (Hylobates agilis), in which the [AGC]n upstream sequence (GGAA) is replaced by a GA dinucleotide. The number of [AGC]n in the examined species ranged between 7 (gorilla) and 13 repeats (owl monkeys), with a polymorphism informative content (PIC) similar to that observed in humans. These results indicate that the 3{prime}UTR [AGC] repeat within the MT-PK gene is evolutionarily conserved, supporting that this region has important regulatory functions.« less

Constraints and consequences of the emergence of amino acid repeats in eukaryotic proteins.

PubMed

Chavali, Sreenivas; Chavali, Pavithra L; Chalancon, Guilhem; de Groot, Natalia Sanchez; Gemayel, Rita; Latysheva, Natasha S; Ing-Simmons, Elizabeth; Verstrepen, Kevin J; Balaji, Santhanam; Babu, M Madan

2017-09-01

Proteins with amino acid homorepeats have the potential to be detrimental to cells and are often associated with human diseases. Why, then, are homorepeats prevalent in eukaryotic proteomes? In yeast, homorepeats are enriched in proteins that are essential and pleiotropic and that buffer environmental insults. The presence of homorepeats increases the functional versatility of proteins by mediating protein interactions and facilitating spatial organization in a repeat-dependent manner. During evolution, homorepeats are preferentially retained in proteins with stringent proteostasis, which might minimize repeat-associated detrimental effects such as unregulated phase separation and protein aggregation. Their presence facilitates rapid protein divergence through accumulation of amino acid substitutions, which often affect linear motifs and post-translational-modification sites. These substitutions may result in rewiring protein interaction and signaling networks. Thus, homorepeats are distinct modules that are often retained in stringently regulated proteins. Their presence facilitates rapid exploration of the genotype-phenotype landscape of a population, thereby contributing to adaptation and fitness.

Evolution and function of CAG/polyglutamine repeats in protein–protein interaction networks

PubMed Central

Schaefer, Martin H.; Wanker, Erich E.; Andrade-Navarro, Miguel A.

2012-01-01

Expanded runs of consecutive trinucleotide CAG repeats encoding polyglutamine (polyQ) stretches are observed in the genes of a large number of patients with different genetic diseases such as Huntington's and several Ataxias. Protein aggregation, which is a key feature of most of these diseases, is thought to be triggered by these expanded polyQ sequences in disease-related proteins. However, polyQ tracts are a normal feature of many human proteins, suggesting that they have an important cellular function. To clarify the potential function of polyQ repeats in biological systems, we systematically analyzed available information stored in sequence and protein interaction databases. By integrating genomic, phylogenetic, protein interaction network and functional information, we obtained evidence that polyQ tracts in proteins stabilize protein interactions. This happens most likely through structural changes whereby the polyQ sequence extends a neighboring coiled-coil region to facilitate its interaction with a coiled-coil region in another protein. Alteration of this important biological function due to polyQ expansion results in gain of abnormal interactions, leading to pathological effects like protein aggregation. Our analyses suggest that research on polyQ proteins should shift focus from expanded polyQ proteins into the characterization of the influence of the wild-type polyQ on protein interactions. PMID:22287626

Evolution of genes and repeats in the Nimrod superfamily.

PubMed

Somogyi, Kálmán; Sipos, Botond; Pénzes, Zsolt; Kurucz, Eva; Zsámboki, János; Hultmark, Dan; Andó, István

2008-11-01

The recently identified Nimrod superfamily is characterized by the presence of a special type of EGF repeat, the NIM repeat, located right after a typical CCXGY/W amino acid motif. On the basis of structural features, nimrod genes can be divided into three types. The proteins encoded by Draper-type genes have an EMI domain at the N-terminal part and only one copy of the NIM motif, followed by a variable number of EGF-like repeats. The products of Nimrod B-type and Nimrod C-type genes (including the eater gene) have different kinds of N-terminal domains, and lack EGF-like repeats but contain a variable number of NIM repeats. Draper and Nimrod C-type (but not Nimrod B-type) proteins carry a transmembrane domain. Several members of the superfamily were claimed to function as receptors in phagocytosis and/or binding of bacteria, which indicates an important role in the cellular immunity and the elimination of apoptotic cells. In this paper, the evolution of the Nimrod superfamily is studied with various methods on the level of genes and repeats. A hypothesis is presented in which the NIM repeat, along with the EMI domain, emerged by structural reorganizations at the end of an EGF-like repeat chain, suggesting a mechanism for the formation of novel types of repeats. The analyses revealed diverse evolutionary patterns in the sequences containing multiple NIM repeats. Although in the Nimrod B and Nimrod C proteins show characteristics of independent evolution, many internal NIM repeats in Eater sequences seem to have undergone concerted evolution. An analysis of the nimrod genes has been performed using phylogenetic and other methods and an evolutionary scenario of the origin and diversification of the Nimrod superfamily is proposed. Our study presents an intriguing example how the evolution of multigene families may contribute to the complexity of the innate immune response.

Plasmodium cysteine repeat modular proteins 1-4: complex proteins with roles throughout the malaria parasite life cycle.

PubMed

Thompson, Joanne; Fernandez-Reyes, Delmiro; Sharling, Lisa; Moore, Sally G; Eling, Wijnand M; Kyes, Sue A; Newbold, Christopher I; Kafatos, Fotis C; Janse, Chris J; Waters, Andrew P

2007-06-01

The Cysteine Repeat Modular Proteins (PCRMP1-4) of Plasmodium, are encoded by a small gene family that is conserved in malaria and other Apicomplexan parasites. They are very large, predicted surface proteins with multipass transmembrane domains containing motifs that are conserved within families of cysteine-rich, predicted surface proteins in a range of unicellular eukaryotes, and a unique combination of protein-binding motifs, including a >100 kDa cysteine-rich modular region, an epidermal growth factor-like domain and a Kringle domain. PCRMP1 and 2 are expressed in life cycle stages in both the mosquito and vertebrate. They colocalize with PfEMP1 (P. falciparum Erythrocyte Membrane Antigen-1) during its export from P. falciparum blood-stage parasites and are exposed on the surface of haemolymph- and salivary gland-sporozoites in the mosquito, consistent with a role in host tissue targeting and invasion. Gene disruption of pcrmp1 and 2 in the rodent malaria model, P. berghei, demonstrated that both are essential for transmission of the parasite from the mosquito to the mouse and has established their discrete and important roles in sporozoite targeting to the mosquito salivary gland. The unprecedented expression pattern and structural features of the PCRMPs thus suggest a variety of roles mediating host-parasite interactions throughout the parasite life cycle.

Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

PubMed Central

Neuwald, A. F.; Liu, J. S.; Lawrence, C. E.

1995-01-01

The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles. PMID:8520488

Stringent Control of NFE2L3 (Nuclear Factor, Erythroid 2-Like 3; NRF3) Protein Degradation by FBW7 (F-box/WD Repeat-containing Protein 7) and Glycogen Synthase Kinase 3 (GSK3)*

PubMed Central

Kannan, Meenakshi B.; Dodard-Friedman, Isadore; Blank, Volker

2015-01-01

The NFE2L3 transcription factor has been implicated in various cellular processes, including carcinogenesis, stress response, differentiation, and inflammation. Previously it has been shown that NFE2L3 has a rapid turnover and is stabilized by proteasomal inhibitors. The mechanisms regulating the degradation of this protein have not been investigated. Here we report ubiquitination of NFE2L3 and demonstrate that F-box/WD repeat-containing protein 7 (FBW7 or FBWX7), a component of Skp1, Cullin 1, F-box containing complex (SCF)-type E3 ligase, is the E3 ligase mediating the degradation of NFE2L3. We showed that FBW7 interacts with NFE2L3 and that dimerization of FBW7 is required for the degradation of the transcription factor. We also demonstrate that the kinase glycogen synthase kinase 3 (GSK3) mediates the FBW7-dependent ubiquitination of NFE2L3. We show phosphorylation of NFE2L3 by GSK3 and its significance in the regulation of NFE2L3 by the tumor suppressor FBW7. FBW7 abrogated NFE2L3-mediated repression of the NAD(P)H:quinone oxidoreductase 1 (NQO1) gene antioxidant response element (ARE). Our findings reveal FBW7 and GSK3 as novel regulators of the NFE2L3 transcription factor and a potential mechanism by which FBW7 might regulate detoxification and the cellular response to stress. PMID:26306035

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain

PubMed Central

de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

2014-01-01

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution. PMID:24792163

[Textual research on Guang dong xin yu (New Sayings of Guangdong) quoted in Ben cao gang mu shi yi (Supplements to Compendium of Materia Medica].

PubMed

Zhang, Ruixian; Zhang, Wei; Li, Jian; Liang, Fei

2014-05-01

Altogether 15 terms for Guang dong xin yu (New Sayings of Guangdong) were used in Ben cao gang mu shi yi (Supplements to Compendium of Materia Medica), including Yue yu (Cantonese sayings), Chong yu (Sayings from Insect Drug), Jie yu (Sayings from Crustacean Drug), Xin yu (New Sayings), Yue hai xiang yu (Fragrant Sayings from Cantonese Region), Yue zhi mu yu (Sayings from Plants in Cantonese Annals), Guang dong suo yu (Trivial Sayings from Guangdong), Yue shan lu (Records of Cantonese Mountains), Yue lu (Cantonese Records), Jiao guang lu (Joint Guangdong Records), Yue cao zhi (Records of Cantonese Grasses), Guang guo lu (Records of Guangdong Fruits), Nan yue suo ji (Trivial Records of Southern Canton), Guang zhi (Guangdong Records), Yue zhi (Cantonese Records) etc. dealing with 57 sorts of drugs (with individual overlapping ones), the author of Xin yu was Qu Dajun, a surviving fogy of the Ming Dynasty actively involved in the activities to restore the old dynasty and resist the Qing Dynasty, and was persecuted in the literary inquisition in which his works were burnt so that Zhao Xuemin, when quoting his texts, had to go in a roundabout way.

Sel1 repeat protein LpnE is a Legionella pneumophila virulence determinant that influences vacuolar trafficking.

PubMed

Newton, Hayley J; Sansom, Fiona M; Dao, Jenny; McAlister, Adrian D; Sloan, Joan; Cianciotto, Nicholas P; Hartland, Elizabeth L

2007-12-01

The environmental pathogen Legionella pneumophila possesses five proteins with Sel1 repeats (SLRs) from the tetratricopeptide repeat protein family. Three of these proteins, LpnE, EnhC, and LidL, have been implicated in the ability of L. pneumophila to efficiently establish infection and/or manipulate host cell trafficking events. Previously, we showed that LpnE is important for L. pneumophila entry into macrophages and epithelial cells. In further virulence studies here, we show that LpnE is also required for efficient infection of Acanthamoeba castellanii by L. pneumophila and for replication of L. pneumophila in the lungs of A/J mice. In addition, we found that the role of LpnE in host cell invasion is dependent on the eight SLR regions of the protein. A truncated form of LpnE lacking the two C-terminal SLR domains was unable to complement the invasion defect of an lpnE mutant of L. pneumophila 130b in both the A549 and THP-1 cell lines. The lpnE mutant displayed impaired avoidance of LAMP-1 association, suggesting that LpnE influenced trafficking of the L. pneumophila vacuole, similar to the case for EnhC and LidL. We also found that LpnE was present in L. pneumophila culture supernatants and that its export was independent of both the Lsp type II secretion system and the Dot/Icm type IV secretion system. The fact that LpnE was exported suggested that the protein may interact with a eukaryotic protein. Using LpnE as bait, we screened a HeLa cell cDNA library for interacting partners, using the yeast two-hybrid system. Examination of the protein-protein interaction between LpnE and a eukaryotic protein, obscurin-like protein 1, suggested that LpnE can interact with eukaryotic proteins containing immunoglobulin-like folds via the SLR regions. This investigation has further characterized the contribution of LpnE to L. pneumophila virulence and, more specifically, the importance of the SLR regions to LpnE function.

Novel protein domains and repeats in Drosophila melanogaster: insights into structure, function, and evolution.

PubMed

Ponting, C P; Mott, R; Bork, P; Copley, R R

2001-12-01

Sequence database searching methods such as BLAST, are invaluable for predicting molecular function on the basis of sequence similarities among single regions of proteins. Searches of whole databases however, are not optimized to detect multiple homologous regions within a single polypeptide. Here we have used the prospero algorithm to perform self-comparisons of all predicted Drosophila melanogaster gene products. Predicted repeats, and their homologs from all species, were analyzed further to detect hitherto unappreciated evolutionary relationships. Results included the identification of novel tandem repeats in the human X-linked retinitis pigmentosa type-2 gene product, repeated segments in cystinosin, associated with a defect in cystine transport, and 'nested' homologous domains in dysferlin, whose gene is mutated in limb girdle muscular dystrophy. Novel signaling domain families were found that may regulate the microtubule-based cytoskeleton and ubiquitin-mediated proteolysis, respectively. Two families of glycosyl hydrolases were shown to contain internal repetitions that hint at their evolution via a piecemeal, modular approach. In addition, three examples of fruit fly genes were detected with tandem exons that appear to have arisen via internal duplication. These findings demonstrate how completely sequenced genomes can be exploited to further understand the relationships between molecular structure, function, and evolution.

High-Pressure NMR and SAXS Reveals How Capping Modulates Folding Cooperativity of the pp32 Leucine-rich Repeat Protein.

PubMed

Zhang, Yi; Berghaus, Melanie; Klein, Sean; Jenkins, Kelly; Zhang, Siwen; McCallum, Scott A; Morgan, Joel E; Winter, Roland; Barrick, Doug; Royer, Catherine A

2018-04-27

Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine-rich repeat protein, pp32, by monitoring the pressure- and urea-induced unfolding of an N-terminal capping motif (N-cap) deletion mutant, pp32-∆N-cap, and a C-terminal capping motif destabilization mutant pp32-Y131F/D146L, using residue-specific NMR and small-angle X-ray scattering. Destabilization of the C-terminal capping motif resulted in higher cooperativity for the unfolding transition compared to wild-type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-cap led to strong deviation from two-state unfolding. In both urea- and pressure-induced unfolding, residues in repeats 1-3 of pp32-ΔN-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-ΔN-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-ΔN-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-ΔN-cap variant arise from their distinct mechanisms of action. Copyright © 2018 Elsevier Ltd. All rights reserved.

The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA complex

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

Wang, Ruiying; Zheng, Han; Preamplume, Gan

The repeat-associated mysterious proteins (RAMPs) comprise the most abundant family of proteins involved in prokaryotic immunity against invading genetic elements conferred by the clustered regularly interspaced short palindromic repeat (CRISPR) system. Cas6 is one of the first characterized RAMP proteins and is a key enzyme required for CRISPR RNA maturation. Despite a strong structural homology with other RAMP proteins that bind hairpin RNA, Cas6 distinctly recognizes single-stranded RNA. Previous structural and biochemical studies show that Cas6 captures the 5' end while cleaving the 3' end of the CRISPR RNA. Here, we describe three structures and complementary biochemical analysis of amore » noncatalytic Cas6 homolog from Pyrococcus horikoshii bound to CRISPR repeat RNA of different sequences. Our study confirms the specificity of the Cas6 protein for single-stranded RNA and further reveals the importance of the bases at Positions 5-7 in Cas6-RNA interactions. Substitutions of these bases result in structural changes in the protein-RNA complex including its oligomerization state.« less

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain.

PubMed

de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

2014-06-01

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Repeated immobilization stress increases uncoupling protein 1 expression and activity in Wistar rats.

PubMed

Gao, Bihu; Kikuchi-Utsumi, Kazue; Ohinata, Hiroshi; Hashimoto, Masaaki; Kuroshima, Akihiro

2003-06-01

Repeat immobilization-stressed rats are leaner and have improved cold tolerance due to enhancement of brown adipose tissue (BAT) thermogenesis. This process likely involves stress-induced sympathetic nervous system activation and adrenocortical hormone release, which dynamically enhances and suppresses uncoupling protein 1 (UCP1) function, respectively. To investigate whether repeated immobilization influences UCP1 thermogenic properties, we assessed UCP1 mRNA, protein expression, and activity (GDP binding) in BAT from immobilization-naive or repeatedly immobilized rats (3 h daily for 4 weeks) and sham operated or adrenalectomized (ADX) rats. UCP1 properties were assessed before (basal) and after exposure to 3 h of acute immobilization. Basal levels of GDP binding and UCP1 expression was significantly increased (140 and 140%) in the repeated immobilized group. Acute immobilization increased GDP binding in both naive (180%) and repeated immobilized groups (220%) without changing UCP1 expression. In ADX rats, basal GDP binding and UCP1 gene expression significantly increased (140 and 110%), and acute immobilization induced further increase. These data demonstrate that repeated immobilization resulted in enhanced UCP1 function, suggesting that enhanced BAT thermogenesis contributes to lower body weight gain through excess energy loss and an improved ability to maintain body temperature during cold exposure.

Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

PubMed Central

Zadravec, Petra; Štrukelj, Borut

2015-01-01

Safety and probiotic properties make lactic acid bacteria (LAB) attractive hosts for surface display of heterologous proteins. Protein display on nonrecombinant microorganisms is preferred for therapeutic and food applications due to regulatory requirements. We displayed two designed ankyrin repeat proteins (DARPins), each possessing affinity for the Fc region of human IgG, on the surface of Lactococcus lactis by fusing them to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA, containing lysine motif (LysM) repeats. Growth medium containing a secreted fusion protein was used to test its heterologous binding to 10 strains of species of the genus Lactobacillus, using flow cytometry, whole-cell enzyme-linked immunosorbent assay (ELISA), and fluorescence microscopy. The fusion proteins bound to the surfaces of all lactobacilli; however, binding to the majority of bacteria was only 2- to 5-fold stronger than that of the control. Lactobacillus salivarius ATCC 11741 demonstrated exceptionally strong binding (32- to 55-fold higher than that of the control) and may therefore be an attractive host for nonrecombinant surface display. Genomic comparison of the species indicated the exopolysaccharides of Lb. salivarius as a possible reason for the difference. Additionally, a 15-fold concentration-dependent increase in nonrecombinant surface display on L. lactis was demonstrated by growing bacteria with sublethal concentrations of the antibiotics chloramphenicol and erythromycin. Nonrecombinant surface display on LAB, based on LysM repeats, was optimized by selecting Lactobacillus salivarius ATCC 11741 as the optimal host and by introducing antibiotics as additives for increasing surface display on L. lactis. Additionally, effective display of DARPins on the surfaces of nonrecombinant LAB has opened up several new therapeutic possibilities. PMID:25576617

A Standardized Chinese Herbal Decoction, Kai-Xin-San, Restores Decreased Levels of Neurotransmitters and Neurotrophic Factors in the Brain of Chronic Stress-Induced Depressive Rats

PubMed Central

Zhu, Kevin Yue; Mao, Qing-Qiu; Ip, Siu-Po; Choi, Roy Chi-Yan; Dong, Tina Ting-Xia; Lau, David Tai-Wai; Tsim, Karl Wah-Keung

2012-01-01

Kai-xin-san (KXS), a Chinese herbal decoction being prescribed by Sun Simiao in Beiji Qianjin Yaofang about 1400 years ago, contains Ginseng Radix et Rhizoma, Polygalae Radix, Acori tatarinowii Rhizoma, and Poria. KXS has been used to treat stress-related psychiatric disease with the symptoms of depression and forgetfulness in ancient China until today. However, the mechanism of its antidepression action is still unknown. Here, the chronic mild-stress-(CMS-) induced depressive rats were applied in exploring the action mechanisms of KXS treatment. Daily intragastric administration of KXS for four weeks significantly alleviated the CMS-induced depressive symptoms displayed by enhanced sucrose consumption. In addition, the expressions of those molecular bio-markers relating to depression in rat brains were altered by the treatment of KXS. These KXS-regulated brain biomarkers included: (i) the levels of dopamine, norepinephrine, and serotonin (ii) the transcript levels of proteins relating to neurotransmitter metabolism; (iii) the transcript levels of neurotrophic factors and their receptors. The results suggested that the anti-depressant-like action of KXS might be mediated by an increase of neurotransmitters and expression of neurotrophic factors and its corresponding receptors in the brain. Thus, KXS could serve as alternative medicine, or health food supplement, for patients suffering from depression. PMID:22973399

Immunogenicity of a recombinant fusion protein of tandem repeat epitopes of foot-and-mouth disease virus type Asia 1 for guinea pigs.

PubMed

Zhang, Q; Yang, Y Q; Zhang, Z Y; Li, L; Yan, W Y; Jiang, W J; Xin, A G; Lei, C X; Zheng, Z X

2002-01-01

In this study, the sequences of capsid protein VPI regions of YNAs1.1 and YNAs1.2 isolates of foot-and-mouth disease virus (FMDV) were analyzed and a peptide containing amino acids (aa) 133-158 of VP1 and aa 20-34 of VP4 of FMDV type Asia I was assumed to contain B and T cell epitopes, because it is hypervariable and includes a cell attachment site RGD located in the G-H loop. The DNA fragments encoding aa 133-158 of VP1 and aa 20-34 of VP4 of FMDV type Asia 1 were chemically synthesized and ligated into a tandem repeat of aa 133-158-20 approximately 34-133-158. In order to enhance its immunogenicity, the tandem repeat was inserted downstream of the beta-galactosidase gene in the expression vector pWR590. This insertion yielded a recombinant expression vector pAS1 encoding the fusion protein. The latter reacted with sera from FMDV type Asia 1-infected animals in vitro and elicited high levels of neutralizing antibodies in guinea pigs. The T cell proliferation in immunized animals increased following stimulation with the fusion protein. It is reported for the first time that a recombinant fusion protein vaccine was produced using B and T cell epitopes of FMDV type Asia 1 and that this fusion protein was immunogenic. The fusion protein reported here can serve as a candidate of fusion epitopes for design of a vaccine against FMDV type Asia 1.

Antihypertensive activity of transgenic rice seed containing an 18-repeat novokinin peptide localized in the nucleolus of endosperm cells.

PubMed

Wakasa, Yuhya; Zhao, Hui; Hirose, Sakiko; Yamauchi, Daiki; Yamada, Yuko; Yang, Lijun; Ohinata, Kousaku; Yoshikawa, Masaaki; Takaiwa, Fumio

2011-09-01

Novokinin (Arg-Pro-Leu-Lys-Pro-Trp, RPLKPW) is a new potent antihypertensive peptide based on the sequence of ovokinin (2-7) derived from ovalbumin. We previously generated transgenic rice seeds in which eight novokinin were fused to storage protein glutelins (GluA2 and GluC) for expression. Oral administration of these seeds to spontaneously hypertensive rats (SHRs) reduced systolic blood pressures at a dose of 1 g seed/kg of SHR. Here, 10- or 18-tandem repeats of novokinin with an endoplasmic reticulum (ER) retention signal (Lys-Asp-Glu-Leu, KDEL) at the C terminus were directly expressed in rice under the control of the glutelin promoter containing its signal peptide. Only small amounts of the 18-repeat novokinin accumulated, and it was unexpectedly deposited in the nucleolus. This abnormal intracellular localization was explained by an endogenous signal for nuclear localization. The GFP reporter protein fused to this sequence targeted to nuclei by a transient assay using onion epidermal cells. Transgenic seed expressing the 18-repeat novokinin exhibited significantly higher antihypertensive activity after a single oral dose to SHR even at one-quarter the amount (0.25 g/kg) of the transgenic rice seed expressing the fusion construct; though, its novokinin content was much lower (1/5). Furthermore, in a long-term administration for 5 weeks, even a smaller dose (0.0625 g/kg) of transgenic seeds could confer antihypertensive activity. This high antihypertensive activity may be attributed to differences in digestibility of expressed products by gastrointestinal enzymes and the unique intracellular localization. These results indicate that accumulation of novokinin as a tandemly repeated structure in transgenic rice is more effective than as a fusion-type structure. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology and Blackwell Publishing Ltd.

Telomeric repeat-containing RNA (TERRA) related to polycystic ovary syndrome (PCOS).

PubMed

Wang, Caiqin; Shen, Fengxian; Zhu, Yuning; Fang, Yuying; Lu, Shiming

2017-04-01

Telomeric repeat-containing RNA (TERRA) participates in the regulation of telomere length, and leucocyte telomere length (LTL) plays an important role in the pathophysiology of polycystic ovary syndrome (PCOS), but little is known about the role of TERRA in PCOS. To evaluate the role of TERRA and peripheral blood LTL in PCOS. Forty women with PCOS and 35 healthy women without PCOS were recruited. A prospective case-control study was performed. RNA fluorescence in situ hybridization (FISH) was used to detect TERRA expression in peripheral blood leucocyte. Quantitative PCR was used to measure TERRA expression and the mean LTL in the PCOS and control groups. We analysed the association between related clinical parameters and the age-adjusted ratio of the telomere repeat length (T/S ratio) or TERRA. Telomeric repeat-containing RNA was expressed in human peripheral blood leucocytes, and the signal was abolished after culture with RNase A. The age-adjusted LTLs were significantly longer in the PCOS group than in the control group (P < 0·01). The age-adjusted TERRA level was significantly lower in the PCOS group than in the control group (P < 0·01). Testosterone (TTE) was related positively to LTL and negatively to TERRA in the PCOS group (r = 0·532, P = 0·002; r = -0·477, P = 0·017). We found TERRA expression in human peripheral blood leucocytes, and LTLs were positively associated with PCOS. TERRA and testosterone play an important role in the LTL regulation in PCOS. © 2016 John Wiley & Sons Ltd.

A Novel Peptide Derived from the Fusion Protein Heptad Repeat Inhibits Replication of Subacute Sclerosing Panencephalitis Virus In Vitro and In Vivo.

PubMed

Watanabe, Masahiro; Hashimoto, Koichi; Abe, Yusaku; Kodama, Eiichi N; Nabika, Ryota; Oishi, Shinya; Ohara, Shinichiro; Sato, Masatoki; Kawasaki, Yukihiko; Fujii, Nobutaka; Hosoya, Mitsuaki

2016-01-01

Subacute sclerosing panencephalitis (SSPE) is a persistent, progressive, and fatal degenerative disease resulting from persistent measles virus (MV) infection of the central nervous system. Most drugs used to treat SSPE have been reported to have limited effects. Therefore, novel therapeutic strategies are urgently required. The SSPE virus, a variant MV strain, differs virologically from wild-type MV strain. One characteristic of the SSPE virus is its defective production of cell-free virus, which leaves cell-to-cell infection as the major mechanism of viral dissemination. The fusion protein plays an essential role in this cell-to-cell spread. It contains two critical heptad repeat regions that form a six-helix bundle in the trimer similar to most viral fusion proteins. In the case of human immunodeficiency virus type-1 (HIV-1), a synthetic peptide derived from the heptad repeat region of the fusion protein enfuvirtide inhibits viral replication and is clinically approved as an anti-HIV-1 agent. The heptad repeat regions of HIV-1 are structurally and functionally similar to those of the MV fusion protein. We therefore designed novel peptides derived from the fusion protein heptad repeat region of the MV and examined their effects on the measles and SSPE virus replication in vitro and in vivo. Some of these synthetic novel peptides demonstrated high antiviral activity against both the measles (Edmonston strain) and SSPE (Yamagata-1 strain) viruses at nanomolar concentrations with no cytotoxicity in vitro. In particular, intracranial administration of one of the synthetic peptides increased the survival rate from 0% to 67% in an SSPE virus-infected nude mouse model.

A Novel Peptide Derived from the Fusion Protein Heptad Repeat Inhibits Replication of Subacute Sclerosing Panencephalitis Virus In Vitro and In Vivo

PubMed Central

Watanabe, Masahiro; Hashimoto, Koichi; Abe, Yusaku; Kodama, Eiichi N.; Nabika, Ryota; Oishi, Shinya; Ohara, Shinichiro; Sato, Masatoki; Kawasaki, Yukihiko; Fujii, Nobutaka; Hosoya, Mitsuaki

2016-01-01

Subacute sclerosing panencephalitis (SSPE) is a persistent, progressive, and fatal degenerative disease resulting from persistent measles virus (MV) infection of the central nervous system. Most drugs used to treat SSPE have been reported to have limited effects. Therefore, novel therapeutic strategies are urgently required. The SSPE virus, a variant MV strain, differs virologically from wild-type MV strain. One characteristic of the SSPE virus is its defective production of cell-free virus, which leaves cell-to-cell infection as the major mechanism of viral dissemination. The fusion protein plays an essential role in this cell-to-cell spread. It contains two critical heptad repeat regions that form a six-helix bundle in the trimer similar to most viral fusion proteins. In the case of human immunodeficiency virus type-1 (HIV-1), a synthetic peptide derived from the heptad repeat region of the fusion protein enfuvirtide inhibits viral replication and is clinically approved as an anti-HIV-1 agent. The heptad repeat regions of HIV-1 are structurally and functionally similar to those of the MV fusion protein. We therefore designed novel peptides derived from the fusion protein heptad repeat region of the MV and examined their effects on the measles and SSPE virus replication in vitro and in vivo. Some of these synthetic novel peptides demonstrated high antiviral activity against both the measles (Edmonston strain) and SSPE (Yamagata-1 strain) viruses at nanomolar concentrations with no cytotoxicity in vitro. In particular, intracranial administration of one of the synthetic peptides increased the survival rate from 0% to 67% in an SSPE virus-infected nude mouse model. PMID:27612283

Potential ex vivo immunomodulatory effects of San-Huang-Xie-Xin-Tang and its component herbs on mice and humans.

PubMed

Li, Chia-Yang; Hou, Yu-Chi; Lee Chao, Pei-Dawn; Shia, Chi-Sheng; Hsu, Ian C; Fang, Shih-Hua

2010-02-03

San-Huang-Xie-Xin-Tang (SHXXT), an important Chinese medicine formula, contains Rhei Rhizoma (RR), Scutellariae Radix (SR) and Coptidis Rhizoma (CR). RR and SR are abundant in anthraquinone and flavonoid polyphenols. Pharmacokinetic study of SHXXT indicated that glucuronides were the predominant forms of polyphenols in rats. As an extension of pharmacokinetic study, the serum metabolites of SHXXT, RR, SR and CR were prepared from rats and quantitated, then the immunomodulation effects were examined by culturing these serum metabolites with murine and human immune cells. The results indicated that the inhibitions on nitric oxide (NO) and cytokine production from mitogen-activated peritoneal macrophages by the serum metabolites of SHXXT, RR, SR and CR were through reducing the protein expression of inducible NO synthase (iNOS) and the IC(50) were 0.8%, 1.5%, 3.0% and 0.8% of their blood concentrations, respectively. In addition, the serum metabolites of SHXXT, RR, SR and CR significantly decreased the ratios of interferon-gamma (IFN-gamma) to interleukin (IL)-4 in mitogen-stimulated mice spleen cells and human peripheral blood mononuclear cells (PBMCs). Moreover, the serum metabolites of SHXXT and SR significantly arrested the mitogen-stimulated mice spleen cells at G2/M stage. In conclusion, the serum metabolites of SHXXT and the component herbs exerted promising modulation activities on the immune functions and the cell cycle distribution of mice and human immune cells. We suggest that SHXXT is a promising remedy for immunomodulation through Th1/Th2 regulation. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

Comparative analysis of the main bioactive components of Xin-Sheng-Hua granule and its single herbs by ultrahigh performance liquid chromatography with tandem mass spectrometry.

PubMed

Pang, Hanqing; Wang, Jun; Tang, Yuping; Xu, Huiqin; Wu, Liang; Jin, Yi; Zhu, Zhenhua; Guo, Sheng; Shi, Xuqin; Huang, Shengliang; Sun, Dazheng; Duan, Jin-Ao

2016-11-01

Xin-Sheng-Hua granule, a representative formula for postpartum hemorrhage, has been used clinically to treat postpartum diseases. Its main bioactive components comprise aromatic acids, phthalides, alkaloids, flavonoids, and gingerols among others. To investigate the changes in main bioactive constituents in its seven single herbs before and after compatibility, a rapid, simple, and sensitive method was developed for comparative analysis of 27 main bioactive components by using ultrahigh-performance liquid chromatography with triple quadrupole electrospray tandem mass spectrometry for the first time. The sufficient separation of 27 target constituents was achieved on a Thermo Scientific Hypersil GOLD column (100 mm × 3 mm, 1.9 μm) within 20 min under the optimized chromatographic conditions. Compared with the theoretical content, the observed content of each analyte showed remarkable differences in Xin-Sheng-Hua granule except thymine, p-coumaric acid, senkyunolide I, senkyunolide H, and ligustilide; the total contents of 27 components increased significantly, and the content variation degrees for the different components were gingerols > flavonoids > aromatic acids > alkaloids > phthalides. The results could provide a good reference for the quality control of Xin-Sheng-Hua granule and might be helpful to interpret the drug interactions based on variation of bioactive components in formulae. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Standardized Chinese Formula Xin-Ke-Shu inhibits the myocardium Ca2+ overloading and metabolic alternations in isoproterenol-induced myocardial infarction rats

PubMed Central

Liu, Yue-Tao; Zhou, Chao; Jia, Hong-Mei; Chang, Xing; Zou, Zhong-Mei

2016-01-01

Xin-Ke-Shu (XKS) is a traditional Chinese patent medicine used for treatment of coronary heart diseases in China. However, its mechanism of action is still unclear. In this paper, the mediation of XKS on the isoproterenol (ISO)-induced myocardial infarction (MI) rat were evaluated based on a tissue-targeted metabonomics in vitro/vivo. The result indicated that twelve metabolic pathways were involved in the therapeutic effect of XKS in vivo, where seven pathways were associated with the Ca2+ overloading mechanism. In agreement with regulation on metabolic variations, XKS markedly reversed the over-expressions of three involved proteins including phospholipase A2 IIA (PLA2 IIA), calcium/calmodulin-dependent protein kinase II (CaMK II) and Pro-Caspase-3. The metabolic regulations of XKS on H9c2 cell also partially confirmed its metabolic effect. These metabolic characteristics in vitro/vivo and western blotting analysis suggested that XKS protected from MI metabolic perturbation major via inhibition of Ca2+ overloading mechanism. Furthermore, 11 active ingredients of XKS exerted steady affinity with the three proteins through the molecular docking study. Our findings indicate that the metabonomics in vitro/vivo combined with western blotting analysis offers the opportunity to gain insight into the comprehensive efficacy of TCMs on the whole metabolic network. PMID:27457884

The crystal structure of a partial mouse Notch-1 ankyrin domain: Repeats 4 through 7 preserve an ankyrin fold

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

Lubman, Olga Y.; Kopan, Raphael; Waksman, Gabriel

Folding and stability of proteins containing ankyrin repeats (ARs) is of great interest because they mediate numerous protein-protein interactions involved in a wide range of regulatory cellular processes. Notch, an ankyrin domain containing protein, signals by converting a transcriptional repression complex into an activation complex. The Notch ANK domain is essential for Notch function and contains seven ARs. Here, we present the 2.2 {angstrom} crystal structure of ARs 4-7 from mouse Notch 1 (m1ANK). These C-terminal repeats were resistant to degradation during crystallization, and their secondary and tertiary structures are maintained in the absence of repeats 1-3. The crystallized fragmentmore » adopts a typical ankyrin fold including the poorly conserved seventh AR, as seen in the Drosophila Notch ANK domain (dANK). The structural preservation and stability of the C-terminal repeats shed a new light onto the mechanism of hetero-oligomeric assembly during Notch-mediated transcriptional activation.« less

ACCA phosphopeptide recognition by the BRCT repeats of BRCA1.

PubMed

Ray, Hind; Moreau, Karen; Dizin, Eva; Callebaut, Isabelle; Venezia, Nicole Dalla

2006-06-16

The tumour suppressor gene BRCA1 encodes a 220 kDa protein that participates in multiple cellular processes. The BRCA1 protein contains a tandem of two BRCT repeats at its carboxy-terminal region. The majority of disease-associated BRCA1 mutations affect this region and provide to the BRCT repeats a central role in the BRCA1 tumour suppressor function. The BRCT repeats have been shown to mediate phospho-dependant protein-protein interactions. They recognize phosphorylated peptides using a recognition groove that spans both BRCT repeats. We previously identified an interaction between the tandem of BRCA1 BRCT repeats and ACCA, which was disrupted by germ line BRCA1 mutations that affect the BRCT repeats. We recently showed that BRCA1 modulates ACCA activity through its phospho-dependent binding to ACCA. To delineate the region of ACCA that is crucial for the regulation of its activity by BRCA1, we searched for potential phosphorylation sites in the ACCA sequence that might be recognized by the BRCA1 BRCT repeats. Using sequence analysis and structure modelling, we proposed the Ser1263 residue as the most favourable candidate among six residues, for recognition by the BRCA1 BRCT repeats. Using experimental approaches, such as GST pull-down assay with Bosc cells, we clearly showed that phosphorylation of only Ser1263 was essential for the interaction of ACCA with the BRCT repeats. We finally demonstrated by immunoprecipitation of ACCA in cells, that the whole BRCA1 protein interacts with ACCA when phosphorylated on Ser1263.

Tandem Repeats in Proteins: Prediction Algorithms and Biological Role.

PubMed

Pellegrini, Marco

2015-01-01

Tandem repetitions in protein sequence and structure is a fascinating subject of research which has been a focus of study since the late 1990s. In this survey, we give an overview on the multi-faceted aspects of research on protein tandem repeats (PTR for short), including prediction algorithms, databases, early classification efforts, mechanisms of PTR formation and evolution, and synthetic PTR design. We also touch on the rather open issue of the relationship between PTR and flexibility (or disorder) in proteins. Detection of PTR either from protein sequence or structure data is challenging due to inherent high (biological) signal-to-noise ratio that is a key feature of this problem. As early in silico analytic tools have been key enablers for starting this field of study, we expect that current and future algorithmic and statistical breakthroughs will have a high impact on the investigations of the biological role of PTR.

Critical thickness of MBE-grown Ga 1-xIn xSb ( x<0.2) on GaSb

NASA Astrophysics Data System (ADS)

Nilsen, T. A.; Breivik, M.; Selvig, E.; Fimland, B. O.

2009-03-01

Several Ga 1-xIn xSb layers, capped with 1 μm of GaSb, were grown on GaSb(0 0 1) substrates by molecular beam epitaxy in a Varian Gen II Modular system using either the conventional sample growth position with substrate rotation, or a tilted sample position with no substrate rotation. The GaInSb layers were examined by X-ray diffraction (XRD) using both symmetrical and asymmetrical reflections. The "tilted sample method" gave a variation of ±25% in thickness of the Ga 1-xIn xSb layers, while the indium (In) content varied by ±10% around the nominal value. The disappearance of thickness fringes in 004 XRD scans was used to determine the onset of relaxation, as determining the in-plane lattice constant for tilted samples was found to be difficult. Determining residual strain in samples grown by the tilted method was likewise found to be very difficult. The critical thickness for several In mole fractions between 5% and 19% was determined and was found to be from 2.2 to 2.7 times higher than predicted by Matthews and Blakeslee (1974) [J. Crystal Growth 27 (1974) 118] but lower than that predicted by People and Bean (1985) [Appl. Phys. Lett. 47 (1985) 322].

Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein

PubMed Central

Oyen, David; Torres, Jonathan L.; Wille-Reece, Ulrike; Ockenhouse, Christian F.; Emerling, Daniel; Glanville, Jacob; Volkmuth, Wayne; Flores-Garcia, Yevel; Zavala, Fidel; Ward, Andrew B.; King, C. Richter; Wilson, Ian A.

2017-01-01

Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development in vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA)3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP–Fab311 complex forming a highly organized helical structure. Together, these structural insights may aid in the design of a next-generation malaria vaccine. PMID:29138320

Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein

DOE PAGES

Oyen, David; Torres, Jonathan L.; Wille-Reece, Ulrike; ...

2017-11-14

Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here in this paper, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development inmore » vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA) 3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP–Fab311 complex forming a highly organized helical structure. Lastly, together, these structural insights may aid in the design of a next-generation malaria vaccine.« less

Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein

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

Oyen, David; Torres, Jonathan L.; Wille-Reece, Ulrike

Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here in this paper, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development inmore » vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA) 3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP–Fab311 complex forming a highly organized helical structure. Lastly, together, these structural insights may aid in the design of a next-generation malaria vaccine.« less

Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B.

PubMed Central

Létoffé, S; Wandersman, C

1992-01-01

Protease B from Erwinia chrysanthemi was shown previously to have a C-terminal secretion signal located downstream of a domain that contains six glycine-rich repeats. This domain is conserved in all known bacterial proteins secreted by the signal peptide-independent pathway. The role of these repeats in the secretion process is controversial. We compared the secretion processes of various heterologous polypeptides fused either directly to the signal or separated from it by the glycine-rich domain. Although the repeats are not involved in the secretion of small truncated protease B carboxy-terminal peptides, they are required for the secretion of higher-molecular-weight fusion proteins. Secretion efficiency was also dependent on the size of the passenger polypeptide. Images PMID:1629152

The concept of the phases ratio control during the formation of composite filamentary nanocrystals xInSe-(1-x)In2O3 on glass substrates

NASA Astrophysics Data System (ADS)

Gasanly, S. A.; Tomaev, V. V.; Stoyanova, T. V.

2017-11-01

The method of vacuum deposition on substrates of glass marks the C-29 series PbSe deposited film and the film In the area 3x3 mm2 and a thickness of ˜1 μm. Films are oxidized in dry air at a temperature of 550 °C. Based on studies by X-ray microanalysis and scanning electron microscopy shows the principal possibility of formation of nanowires xInSe-(1-x)In2O3 on the PbSe/In structure. The results allowed to formulate the concept of the control of phases ratio in the forming nanowires xInSe-(1-x)In2O3 on glass substrates.

HLB1 Is a Tetratricopeptide Repeat Domain-Containing Protein That Operates at the Intersection of the Exocytic and Endocytic Pathways at the TGN/EE in Arabidopsis

DOE PAGES

Sparks, J. Alan; Kwon, Taegun; Renna, Luciana; ...

2016-03-03

The endomembrane system plays essential roles in plant development, but the proteome responsible for its function and organization remains largely uncharacterized in plants. For this study, we identified and characterized the HYPERSENSITIVE TO LATRUNCULIN B1 (HLB1) protein isolated through a forward-genetic screen in Arabidopsis thaliana for mutants with heightened sensitivity to actin-disrupting drugs. HLB1 is a plant-specific tetratricopeptide repeat domain-containing protein of unknown function encoded by a single Arabidopsis gene. HLB1 associated with the trans-Golgi network (TGN)/early endosome (EE) and tracked along filamentous actin, indicating that it could link post-Golgi traffic with the actin cytoskeleton in plants. HLB1 was foundmore » to interact with the ADP-ribosylation-factor guanine nucleotide exchange factor, MIN7/BEN1 (HOPM INTERACTOR7/BREFELDIN A-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1) by coimmunoprecipitation. The min7/ben1 mutant phenocopied the mild root developmental defects and latrunculin B hypersensitivity of hlb1, and analyses of a hlb1/ min7/ben1 double mutant showed that hlb1 and min7/ben1 operate in common genetic pathways. Based on these data, we propose that HLB1 together with MIN7/BEN1 form a complex with actin to modulate the function of the TGN/EE at the intersection of the exocytic and endocytic pathways in plants.« less

Identification of TTAGGG-binding proteins in Neurospora crassa, a fungus with vertebrate-like telomere repeats.

PubMed

Casas-Vila, Núria; Scheibe, Marion; Freiwald, Anja; Kappei, Dennis; Butter, Falk

2015-11-17

To date, telomere research in fungi has mainly focused on Saccharomyces cerevisiae and Schizosaccharomyces pombe, despite the fact that both yeasts have degenerated telomeric repeats in contrast to the canonical TTAGGG motif found in vertebrates and also several other fungi. Using label-free quantitative proteomics, we here investigate the telosome of Neurospora crassa, a fungus with canonical telomeric repeats. We show that at least six of the candidates detected in our screen are direct TTAGGG-repeat binding proteins. While three of the direct interactors (NCU03416 [ncTbf1], NCU01991 [ncTbf2] and NCU02182 [ncTay1]) feature the known myb/homeobox DNA interaction domain also found in the vertebrate telomeric factors, we additionally show that a zinc-finger protein (NCU07846) and two proteins without any annotated DNA-binding domain (NCU02644 and NCU05718) are also direct double-strand TTAGGG binders. We further find two single-strand binders (NCU02404 [ncGbp2] and NCU07735 [ncTcg1]). By quantitative label-free interactomics we identify TTAGGG-binding proteins in Neurospora crassa, suggesting candidates for telomeric factors that are supported by phylogenomic comparison with yeast species. Intriguingly, homologs in yeast species with degenerated telomeric repeats are also TTAGGG-binding proteins, e.g. in S. cerevisiae Tbf1 recognizes the TTAGGG motif found in its subtelomeres. However, there is also a subset of proteins that is not conserved. While a rudimentary core TTAGGG-recognition machinery may be conserved across yeast species, our data suggests Neurospora as an emerging model organism with unique features.

Single Amino Acid Repeats in the Proteome World: Structural, Functional, and Evolutionary Insights

PubMed Central

Kumar, Amitha Sampath; Sowpati, Divya Tej; Mishra, Rakesh K.

2016-01-01

Microsatellites or simple sequence repeats (SSR) are abundant, highly diverse stretches of short DNA repeats present in all genomes. Tandem mono/tri/hexanucleotide repeats in the coding regions contribute to single amino acids repeats (SAARs) in the proteome. While SSRs in the coding region always result in amino acid repeats, a majority of SAARs arise due to a combination of various codons representing the same amino acid and not as a consequence of SSR events. Certain amino acids are abundant in repeat regions indicating a positive selection pressure behind the accumulation of SAARs. By analysing 22 proteomes including the human proteome, we explored the functional and structural relationship of amino acid repeats in an evolutionary context. Only ~15% of repeats are present in any known functional domain, while ~74% of repeats are present in the disordered regions, suggesting that SAARs add to the functionality of proteins by providing flexibility, stability and act as linker elements between domains. Comparison of SAAR containing proteins across species reveals that while shorter repeats are conserved among orthologs, proteins with longer repeats, >15 amino acids, are unique to the respective organism. Lysine repeats are well conserved among orthologs with respect to their length and number of occurrences in a protein. Other amino acids such as glutamic acid, proline, serine and alanine repeats are generally conserved among the orthologs with varying repeat lengths. These findings suggest that SAARs have accumulated in the proteome under positive selection pressure and that they provide flexibility for optimal folding of functional/structural domains of proteins. The insights gained from our observations can help in effective designing and engineering of proteins with novel features. PMID:27893794

Ehrlichia chaffeensis Tandem Repeat Proteins and Ank200 are Type 1 Secretion System Substrates Related to the Repeats-in-Toxin Exoprotein Family

PubMed Central

Wakeel, Abdul; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; McBride, Jere W.

2011-01-01

Ehrlichia chaffeensis has type 1 and 4 secretion systems (T1SS and T4SS), but the substrates have not been identified. Potential substrates include secreted tandem repeat protein (TRP) 47, TRP120, and TRP32, and the ankyrin repeat protein, Ank200, that are involved in molecular host–pathogen interactions including DNA binding and a network of protein–protein interactions with host targets associated with signaling, transcriptional regulation, vesicle trafficking, and apoptosis. In this study we report that E. chaffeensis TRP47, TRP32, TRP120, and Ank200 were not secreted in the Agrobacterium tumefaciens Cre recombinase reporter assay routinely used to identify T4SS substrates. In contrast, all TRPs and the Ank200 proteins were secreted by the Escherichia coli complemented with the hemolysin secretion system (T1SS), and secretion was reduced in a T1SS mutant (ΔTolC), demonstrating that these proteins are T1SS substrates. Moreover, T1SS secretion signals were identified in the C-terminal domains of the TRPs and Ank200, and a detailed bioinformatic analysis of E. chaffeensis TRPs and Ank200 revealed features consistent with those described in the repeats-in-toxins (RTX) family of exoproteins, including glycine- and aspartate-rich tandem repeats, homology with ATP-transporters, a non-cleavable C-terminal T1SS signal, acidic pIs, and functions consistent with other T1SS substrates. Using a heterologous E. coli T1SS, this investigation has identified the first Ehrlichia T1SS substrates supporting the conclusion that the T1SS and corresponding substrates are involved in molecular host–pathogen interactions that contribute to Ehrlichia pathobiology. Further investigation of the relationship between Ehrlichia TRPs, Ank200, and the RTX exoprotein family may lead to a greater understanding of the importance of T1SS substrates and specific functions of T1SS in the pathobiology of obligately intracellular bacteria. PMID:22919588

Identification of genes containing expanded purine repeats in the human genome and their apparent protective role against cancer.

PubMed

Singh, Himanshu Narayan; Rajeswari, Moganty R

2016-01-01

Purine repeat sequences present in a gene are unique as they have high propensity to form unusual DNA-triple helix structures. Friedreich's ataxia is the only human disease that is well known to be associated with DNA-triplexes formed by purine repeats. The purpose of this study was to recognize the expanded purine repeats (EPRs) in human genome and find their correlation with cancer pathogenesis. We developed "PuRepeatFinder.pl" algorithm to identify non-overlapping EPRs without pyrimidine interruptions in the human genome and customized for searching repeat lengths, n ≥ 200. A total of 1158 EPRs were identified in the genome which followed Wakeby distribution. Two hundred and ninety-six EPRs were found in geneic regions of 282 genes (EPR-genes). Gene clustering of EPR-genes was done based on their cellular function and a large number of EPR-genes were found to be enzymes/enzyme modulators. Meta-analysis of 282 EPR-genes identified only 63 EPR-genes in association with cancer, mostly in breast, lung, and blood cancers. Protein-protein interaction network analysis of all 282 EPR-genes identified proteins including those in cadherins and VEGF. The two observations, that EPRs can induce mutations under malignant conditions and that identification of some EPR-gene products in vital cell signaling-mediated pathways, together suggest the crucial role of EPRs in carcinogenesis. The new link between EPR-genes and their functionally interacting proteins throws a new dimension in the present understanding of cancer pathogenesis and can help in planning therapeutic strategies. Validation of present results using techniques like NGS is required to establish the role of the EPR genes in cancer pathology.

The evolution and function of protein tandem repeats in plants.

PubMed

Schaper, Elke; Anisimova, Maria

2015-04-01

Sequence tandem repeats (TRs) are abundant in proteomes across all domains of life. For plants, little is known about their distribution or contribution to protein function. We exhaustively annotated TRs and studied the evolution of TR unit variations for all Ensembl plants. Using phylogenetic patterns of TR units, we detected conserved TRs with unit number and order preserved during evolution, and those TRs that have diverged via recent TR unit gains/losses. We correlated the mode of evolution of TRs to protein function. TR number was strongly correlated with proteome size, with about one-half of all TRs recognized as common protein domains. The majority of TRs have been highly conserved over long evolutionary distances, some since the separation of red algae and green plants c. 1.6 billion yr ago. Conversely, recurrent recent TR unit mutations were rare. Our results suggest that the first TRs by far predate the first plants, and that TR appearance is an ongoing process with similar rates across the plant kingdom. Interestingly, the few detected highly mutable TRs might provide a source of variation for rapid adaptation. In particular, such TRs are enriched in leucine-rich repeats (LRRs) commonly found in R genes, where TR unit gain/loss may facilitate resistance to emerging pathogens. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Genomic Repeat Abundances Contain Phylogenetic Signal

PubMed Central

Dodsworth, Steven; Chase, Mark W.; Kelly, Laura J.; Leitch, Ilia J.; Macas, Jiří; Novák, Petr; Piednoël, Mathieu; Weiss-Schneeweiss, Hanna; Leitch, Andrew R.

2015-01-01

A large proportion of genomic information, particularly repetitive elements, is usually ignored when researchers are using next-generation sequencing. Here we demonstrate the usefulness of this repetitive fraction in phylogenetic analyses, utilizing comparative graph-based clustering of next-generation sequence reads, which results in abundance estimates of different classes of genomic repeats. Phylogenetic trees are then inferred based on the genome-wide abundance of different repeat types treated as continuously varying characters; such repeats are scattered across chromosomes and in angiosperms can constitute a majority of nuclear genomic DNA. In six diverse examples, five angiosperms and one insect, this method provides generally well-supported relationships at interspecific and intergeneric levels that agree with results from more standard phylogenetic analyses of commonly used markers. We propose that this methodology may prove especially useful in groups where there is little genetic differentiation in standard phylogenetic markers. At the same time as providing data for phylogenetic inference, this method additionally yields a wealth of data for comparative studies of genome evolution. PMID:25261464

Collagen triple helix repeat containing-1 promotes pancreatic cancer progression by regulating migration and adhesion of tumor cells.

PubMed

Park, Eun Hye; Kim, Seokho; Jo, Ji Yoon; Kim, Su Jin; Hwang, Yeonsil; Kim, Jin-Man; Song, Si Young; Lee, Dong-Ki; Koh, Sang Seok

2013-03-01

Collagen triple helix repeat containing-1 (CTHRC1) is a secreted protein involved in vascular remodeling, bone formation and developmental morphogenesis. CTHRC1 has recently been shown to be expressed in human cancers such as breast cancer and melanoma. In this study, we show that CTHRC1 is highly expressed in human pancreatic cancer tissues and plays a role in the progression and metastasis of the disease. CTHRC1 promoted primary tumor growth and metastatic spread of cancer cells to distant organs in orthotopic xenograft tumor mouse models. Overexpression of CTHRC1 in cancer cells resulted in increased motility and adhesiveness, whereas these cellular activities were diminished by down-regulation of the protein. CTHRC1 activated several key signaling molecules, including Src, focal adhesion kinase, paxillin, mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase and Rac1. Treatment with chemical inhibitors of Src, MEK or Rac1 and expression of dominant-negative Rac1 attenuated CTHRC1-induced cell migration and adhesion. Collectively, our results suggest that CTHRC1 has a role in pancreatic cancer progression and metastasis by regulating migration and adhesion activities of cancer cells.

A Bromodomain-Containing Protein from Tomato Specifically Binds Potato Spindle Tuber Viroid RNA In Vitro and In Vivo

PubMed Central

Martínez de Alba, Angel Emilio; Sägesser, Rudolf; Tabler, Martin; Tsagris, Mina

2003-01-01

For the identification of RNA-binding proteins that specifically interact with potato spindle tuber viroid (PSTVd), we subjected a tomato cDNA expression library prepared from viroid-infected leaves to an RNA ligand screening procedure. We repeatedly identified cDNA clones that expressed a protein of 602 amino acids. The protein contains a bromodomain and was termed viroid RNA-binding protein 1 (VIRP1). The specificity of interaction of VIRP1 with viroid RNA was studied by different methodologies, which included Northwestern blotting, plaque lift, and electrophoretic mobility shift assays. VIRP1 interacted strongly and specifically with monomeric and oligomeric PSTVd positive-strand RNA transcripts. Other RNAs, for example, U1 RNA, did not bind to VIRP1. Further, we could immunoprecipitate complexes from infected tomato leaves that contained VIRP1 and viroid RNA in vivo. Analysis of the protein sequence revealed that VIRP1 is a member of a newly identified family of transcriptional regulators associated with chromatin remodeling. VIRP1 is the first member of this family of proteins, for which a specific RNA-binding activity is shown. A possible role of VIRP1 in viroid replication and in RNA mediated chromatin remodeling is discussed. PMID:12915580

Human mismatch repair protein hMutLα is required to repair short slipped-DNAs of trinucleotide repeats.

PubMed

Panigrahi, Gagan B; Slean, Meghan M; Simard, Jodie P; Pearson, Christopher E

2012-12-07

Mismatch repair (MMR) is required for proper maintenance of the genome by protecting against mutations. The mismatch repair system has also been implicated as a driver of certain mutations, including disease-associated trinucleotide repeat instability. We recently revealed a requirement of hMutSβ in the repair of short slip-outs containing a single CTG repeat unit (1). The involvement of other MMR proteins in short trinucleotide repeat slip-out repair is unknown. Here we show that hMutLα is required for the highly efficient in vitro repair of single CTG repeat slip-outs, to the same degree as hMutSβ. HEK293T cell extracts, deficient in hMLH1, are unable to process single-repeat slip-outs, but are functional when complemented with hMutLα. The MMR-deficient hMLH1 mutant, T117M, which has a point mutation proximal to the ATP-binding domain, is defective in slip-out repair, further supporting a requirement for hMLH1 in the processing of short slip-outs and possibly the involvement of hMHL1 ATPase activity. Extracts of hPMS2-deficient HEC-1-A cells, which express hMLH1, hMLH3, and hPMS1, are only functional when complemented with hMutLα, indicating that neither hMutLβ nor hMutLγ is sufficient to repair short slip-outs. The resolution of clustered short slip-outs, which are poorly repaired, was partially dependent upon a functional hMutLα. The joint involvement of hMutSβ and hMutLα suggests that repeat instability may be the result of aberrant outcomes of repair attempts.

Plasmid P1 replication: negative control by repeated DNA sequences.

PubMed Central

Chattoraj, D; Cordes, K; Abeles, A

1984-01-01

The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pieces of the incA fragment that each contain only three repeats destabilize P1 plasmids efficiently. This result makes it unlikely that incA specifies a regulatory product. Our in vivo results suggest that the repeating DNA sequence itself negatively controls replication by titrating a P1-determined protein, RepA, that is essential for replication. Consistent with this hypothesis is the observation that the RepA protein binds to the incA fragment in vitro. Images PMID:6387706

Utilizing protein-lean coproducts from corn containing recombinant pharmaceutical proteins for ethanol production.

PubMed

Paraman, Ilankovan; Moeller, Lorena; Scott, M Paul; Wang, Kan; Glatz, Charles E; Johnson, Lawrence A

2010-10-13

Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were evaluated as a potential feedstock to produce fuel ethanol. The levels of residual r-proteins in the coproduct, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein (r-GFP) and a recombinant subunit vaccine of Escherichia coli enterotoxin (r-LTB), primarily expressed in endosperm, and another two corn lines containing recombinant human collagen (r-CIα1) and r-GFP, primarily expressed in germ, were used as model systems. The kernels were either ground and used for fermentation or dry fractionated to recover germ-rich fractions prior to grinding for fermentation. The finished beers of whole ground kernels and r-protein-spent endosperm solids contained 127-139 and 138-155 g/L ethanol concentrations, respectively. The ethanol levels did not differ among transgenic and normal corn feedstocks, indicating the residual r-proteins did not negatively affect ethanol production. r-Protein extraction and germ removal also did not negatively affect fermentation of the remaining mass. Most r-proteins were inactivated during the mashing process used to prepare corn for fermentation. No functionally active r-GFP or r-LTB proteins were found after fermentation of the r-protein-spent solids; however, a small quantity of residual r-CIα1 was detected in DDGS, indicating that the safety of DDGS produced from transgenic grain for r-protein production needs to be evaluated for each event. Protease treatment during fermentation completely hydrolyzed the residual r-CIα1, and no residual r-proteins were detectable in DDGS.

Advances in the Application of Designed Ankyrin Repeat Proteins (DARPins) as Research Tools and Protein Therapeutics.

PubMed

Boersma, Ykelien L

2018-01-01

Nonimmunoglobulin scaffolds have been developed to overcome the limitations of monoclonal antibodies with regard to stability and size. Of these scaffolds, the class of designed ankyrin repeat proteins (DARPins) has advanced the most in biochemical and biomedical applications. This review focuses on the recent progress in DARPin technology, highlighting the scaffold's potential and possibilities.

Genome-Wide Analyses and Functional Classification of Proline Repeat-Rich Proteins: Potential Role of eIF5A in Eukaryotic Evolution

PubMed Central

Mandal, Ajeet; Mandal, Swati; Park, Myung Hee

2014-01-01

The eukaryotic translation factor, eIF5A has been recently reported as a sequence-specific elongation factor that facilitates peptide bond formation at consecutive prolines in Saccharomyces cerevisiae, as its ortholog elongation factor P (EF-P) does in bacteria. We have searched the genome databases of 35 representative organisms from six kingdoms of life for PPP (Pro-Pro-Pro) and/or PPG (Pro-Pro-Gly)-encoding genes whose expression is expected to depend on eIF5A. We have made detailed analyses of proteome data of 5 selected species, Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster, Mus musculus and Homo sapiens. The PPP and PPG motifs are low in the prokaryotic proteomes. However, their frequencies markedly increase with the biological complexity of eukaryotic organisms, and are higher in newly derived proteins than in those orthologous proteins commonly shared in all species. Ontology classifications of S. cerevisiae and human genes encoding the highest level of polyprolines reveal their strong association with several specific biological processes, including actin/cytoskeletal associated functions, RNA splicing/turnover, DNA binding/transcription and cell signaling. Previously reported phenotypic defects in actin polarity and mRNA decay of eIF5A mutant strains are consistent with the proposed role for eIF5A in the translation of the polyproline-containing proteins. Of all the amino acid tandem repeats (≥3 amino acids), only the proline repeat frequency correlates with functional complexity of the five organisms examined. Taken together, these findings suggest the importance of proline repeat-rich proteins and a potential role for eIF5A and its hypusine modification pathway in the course of eukaryotic evolution. PMID:25364902

Modeling protein homopolymeric repeats: possible polyglutamine structural motifs for Huntington's disease.

PubMed

Lathrop, R H; Casale, M; Tobias, D J; Marsh, J L; Thompson, L M

1998-01-01

We describe a prototype system (Poly-X) for assisting an expert user in modeling protein repeats. Poly-X reduces the large number of degrees of freedom required to specify a protein motif in complete atomic detail. The result is a small number of parameters that are easily understood by, and under the direct control of, a domain expert. The system was applied to the polyglutamine (poly-Q) repeat in the first exon of huntingtin, the gene implicated in Huntington's disease. We present four poly-Q structural motifs: two poly-Q beta-sheet motifs (parallel and antiparallel) that constitute plausible alternatives to a similar previously published poly-Q beta-sheet motif, and two novel poly-Q helix motifs (alpha-helix and pi-helix). To our knowledge, helical forms of polyglutamine have not been proposed before. The motifs suggest that there may be several plausible aggregation structures for the intranuclear inclusion bodies which have been found in diseased neurons, and may help in the effort to understand the structural basis for Huntington's disease.

Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)

PubMed Central

2011-01-01

Background Elevated numbers of regulatory T cells (Tregs) have been implicated in certain cancers. Depletion of Tregs has been shown to increase anti-tumor immunity. Tregs also play a critical role in the suppression of autoimmune responses. The study of Tregs has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated Tregs. However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of Tregs expressing LRRC32. Results Using naturally-occurring freshly isolated Tregs, we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ Tregs are distinct from LRRC32- Tregs with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ Tregs are more potent suppressors than LRRC32- Tregs. Conclusions A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent Treg populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of Tregs and the refinement of immunotherapeutic strategies aimed at targeting these cells. PMID:21615933

Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32).

PubMed

Chan, Derek V; Somani, Ally-Khan; Young, Andrew B; Massari, Jessica V; Ohtola, Jennifer; Sugiyama, Hideaki; Garaczi, Edina; Babineau, Denise; Cooper, Kevin D; McCormick, Thomas S

2011-05-26

Elevated numbers of regulatory T cells (T(regs)) have been implicated in certain cancers. Depletion of T(regs) has been shown to increase anti-tumor immunity. T(regs) also play a critical role in the suppression of autoimmune responses. The study of T(regs) has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated T(regs). However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of T(regs) expressing LRRC32. Using naturally-occurring freshly isolated T(regs), we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ T(regs) are distinct from LRRC32- T(regs) with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ T(regs) are more potent suppressors than LRRC32- T(regs). A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent T(reg) populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of T(regs) and the refinement of immunotherapeutic strategies aimed at targeting these cells.

The BEACH-containing protein WDR81 coordinates p62 and LC3C to promote aggrephagy.

PubMed

Liu, Xuezhao; Li, Yang; Wang, Xin; Xing, Ruxiao; Liu, Kai; Gan, Qiwen; Tang, Changyong; Gao, Zhiyang; Jian, Youli; Luo, Shouqing; Guo, Weixiang; Yang, Chonglin

2017-05-01

Autophagy-dependent clearance of ubiquitinated and aggregated proteins is critical to protein quality control, but the underlying mechanisms are not well understood. Here, we report the essential role of the BEACH (beige and Chediak-Higashi) and WD40 repeat-containing protein WDR81 in eliminating ubiquitinated proteins through autophagy. WDR81 associates with ubiquitin (Ub)-positive protein foci, and its loss causes accumulation of Ub proteins and the autophagy cargo receptor p62. WDR81 interacts with p62, facilitating recognition of Ub proteins by p62. Furthermore, WDR81 interacts with LC3C through canonical LC3-interacting regions in the BEACH domain, promoting LC3C recruitment to ubiquitinated proteins. Inactivation of LC3C or defective autophagy results in accumulation of Ub protein aggregates enriched for WDR81. In mice, WDR81 inactivation causes accumulation of p62 bodies in cortical and striatal neurons in the brain. These data suggest that WDR81 coordinates p62 and LC3C to facilitate autophagic removal of Ub proteins, and provide important insights into CAMRQ2 syndrome, a WDR81-related developmental disorder. © 2017 Liu et al.

The BEACH-containing protein WDR81 coordinates p62 and LC3C to promote aggrephagy

PubMed Central

Xing, Ruxiao; Tang, Changyong; Gao, Zhiyang

2017-01-01

Autophagy-dependent clearance of ubiquitinated and aggregated proteins is critical to protein quality control, but the underlying mechanisms are not well understood. Here, we report the essential role of the BEACH (beige and Chediak–Higashi) and WD40 repeat-containing protein WDR81 in eliminating ubiquitinated proteins through autophagy. WDR81 associates with ubiquitin (Ub)-positive protein foci, and its loss causes accumulation of Ub proteins and the autophagy cargo receptor p62. WDR81 interacts with p62, facilitating recognition of Ub proteins by p62. Furthermore, WDR81 interacts with LC3C through canonical LC3-interacting regions in the BEACH domain, promoting LC3C recruitment to ubiquitinated proteins. Inactivation of LC3C or defective autophagy results in accumulation of Ub protein aggregates enriched for WDR81. In mice, WDR81 inactivation causes accumulation of p62 bodies in cortical and striatal neurons in the brain. These data suggest that WDR81 coordinates p62 and LC3C to facilitate autophagic removal of Ub proteins, and provide important insights into CAMRQ2 syndrome, a WDR81-related developmental disorder. PMID:28404643

Purification of proteins from solutions containing residual host cell proteins via preparative crystallization.

PubMed

Hekmat, Dariusch; Breitschwerdt, Peter; Weuster-Botz, Dirk

2015-09-01

To investigate quantitatively and reproducibly a scalable, preparative crystallization method in novel stirred tanks using three different protein solutions containing residual microbial host cell proteins (HCP). Lysozyme from solutions being spiked with up to 15% host cell proteins (HCP) (corresponding to 176,500 ppm) was crystallized within a 2.4-4.6 h at 93.7% yield using NaCl and glycerol. Lipase was crystallized under comparable conditions using NaCl and a mixture of two polyethylene glycols (PEG). Enhanced green fluorescent protein (eGFP) was overexpressed in E. coli yielding a solution containing 23% target protein. Residual HCP content after pre-treatment was 7-16%. eGFP was crystallized from these solutions within 1.75-4 h at 88.7% step yield using ethanol and the same mixture of two PEG as in the case of lipase. HCP contained in the solvent channels of the protein crystals could be removed by diffusive washing yielding final purities at or above 99%. Preparative crystallization can be carried out with fast kinetics and high yields from solutions containing residual impurities and may represent an attractive alternative purification method compared to preparative chromatography, especially at large production scales.

Topological frustration in βα-repeat proteins: sequence diversity modulates the conserved folding mechanisms of α/β/α sandwich proteins

PubMed Central

Hills, Ronald D.; Kathuria, Sagar V.; Wallace, Louise A.; Day, Iain J.; Brooks, Charles L.; Matthews, C. Robert

2010-01-01

The thermodynamic hypothesis of Anfinsen postulates that structures and stabilities of globular proteins are determined by their amino acid sequences. Chain topology, however, is known to influence the folding reaction, in that motifs with a preponderance of local interactions typically fold more rapidly than those with a larger fraction of non-local interactions. Together, the topology and sequence can modulate the energy landscape and influence the rate at which the protein folds to the native conformation. To explore the relationship of sequence and topology in the folding of βα–repeat proteins, which are dominated by local interactions, a combined experimental and simulation analysis was performed on two members of the flavodoxin-like, α/β/α sandwich fold. Spo0F and the N-terminal receiver domain of NtrC (NT-NtrC) have similar topologies but low sequence identity, enabling a test of the effects of sequence on folding. Experimental results demonstrated that both response-regulator proteins fold via parallel channels through highly structured sub-millisecond intermediates before accessing their cis prolyl peptide bond-containing native conformations. Global analysis of the experimental results preferentially places these intermediates off the productive folding pathway. Sequence-sensitive Gō-model simulations conclude that frustration in the folding in Spo0F, corresponding to the appearance of the off-pathway intermediate, reflects competition for intra-subdomain van der Waals contacts between its N- and C-terminal subdomains. The extent of transient, premature structure appears to correlate with the number of isoleucine, leucine and valine (ILV) side-chains that form a large sequence-local cluster involving the central β-sheet and helices α2, α3 and α4. The failure to detect the off-pathway species in the simulations of NT-NtrC may reflect the reduced number of ILV side-chains in its corresponding hydrophobic cluster. The location of the hydrophobic

Leptospira borgpetersenii hybrid leucine-rich repeat protein: Cloning and expression, immunogenic identification and molecular docking evaluation.

PubMed

Sritrakul, Tepyuda; Nitipan, Supachai; Wajjwalku, Worawidh; La-Ard, Anchalee; Suphatpahirapol, Chattip; Petkarnjanapong, Wimol; Ongphiphadhanakul, Boonsong; Prapong, Siriwan

2017-11-01

Leptospirosis is an important zoonotic disease, and the major outbreak of this disease in Thailand in 1999 was due largely to the Leptospira borgpetersenii serovar Sejroe. Identification of the leucine-rich repeat (LRR) LBJ_2271 protein containing immunogenic epitopes and the discovery of the LBJ_2271 ortholog in Leptospira serovar Sejroe, KU_Sej_R21_2271, led to further studies of the antigenic immune properties of KU_Sej_LRR_2271. The recombinant hybrid (rh) protein was created and expressed from a hybrid PCR fragment of KU_Sej_R21_2271 fused with DNA encoding the LBJ_2271 signal sequence for targeting protein as a membrane-anchoring protein. The fusion DNA was cloned into pET160/GW/D-TOPO® to form the pET160_hKU_R21_2271 plasmid. The plasmid was used to express the rhKU_Sej_LRR_2271 protein in Escherichia coli BL21 Star™ (DE3). The expressed protein was immunologically detected by Western blotting and immunoreactivity detection with hyperimmune sera, T cell epitope prediction by HLA allele and epitope peptide binding affinity, and potential T cell reactivity analysis. The immunogenic epitopes of the protein were evaluated and verified by HLA allele and epitope peptide complex structure molecular docking. Among fourteen best allele epitopes of this protein, binding affinity values of 12 allele epitopes remained unchanged compared to LBJ_2271. Two epitopes for alleles HLA-A0202 and -A0301 had higher IC 50 values, while T cell reactivity values of these peptides were better than values from LBJ_2271 epitopes. Eight of twelve epitope peptides had positive T-cell reactivity scores. Although the molecular docking of two epitopes, 3FPLLKEFLV11/47FPLLKEFLV55 and 50KLSTVPEGV58, into an HLA-A0202 model revealed a good fit in the docked structures, 50KLSTVPEGV58 and 94KLSTVPEEV102 are still considered as the proteins' best epitopes for allele HLA-A0202. The results of this study showed that rhKU_Sej_LRR_2271 protein contained natural immunological properties that should

Repeated-batch operation of immobilized β-galactosidase inclusion bodies-containing Escherichia coli cell reactor for lactose hydrolysis.

PubMed

Yeon, Ji-Hyeon; Jung, Kyung-Hwan

2011-09-01

In this study, we investigated the performance of an immobilized β-galactosidase inclusion bodies-containing Escherichia coli cell reactor, where the cells were immobilized in alginate beads, which were then used in repeated-batch operations for the hydrolysis of o-nitrophenyl-β-D-galactoside or lactose over the long-term. In particular, in the Tris buffer system, disintegration of the alginate beads was not observed during the operation, which was observed for the phosphate buffer system. The o-nitrophenyl-β-D-galactoside hydrolysis was operated successfully up to about 80 h, and the runs were successfully repeated at least eight times. In addition, hydrolysis of lactose was successfully carried out up to 240 h. Using Western blotting analyses, it was verified that the beta-galactosidase inclusion bodies were sustained in the alginate beads during the repeated-batch operations. Consequently, we experimentally verified that β-galactosidase inclusion bodies-containing Escherichia coli cells could be used in a repeated-batch reactor as a biocatalyst for the hydrolysis of o-nitrophenyl-β-D-galactoside or lactose. It is probable that this approach can be applied to enzymatic synthesis reactions for other biotechnology applications, particularly reactions that require long-term and stable operation.

TMPyP4 porphyrin distorts RNA G-quadruplex structures of the disease-associated r(GGGGCC)n repeat of the C9orf72 gene and blocks interaction of RNA-binding proteins.

PubMed

Zamiri, Bita; Reddy, Kaalak; Macgregor, Robert B; Pearson, Christopher E

2014-02-21

Certain DNA and RNA sequences can form G-quadruplexes, which can affect genetic instability, promoter activity, RNA splicing, RNA stability, and neurite mRNA localization. Amyotrophic lateral sclerosis and frontotemporal dementia can be caused by expansion of a (GGGGCC)n repeat in the C9orf72 gene. Mutant r(GGGGCC)n- and r(GGCCCC)n-containing transcripts aggregate in nuclear foci, possibly sequestering repeat-binding proteins such as ASF/SF2 and hnRNPA1, suggesting a toxic RNA pathogenesis, as occurs in myotonic dystrophy. Furthermore, the C9orf72 repeat RNA was recently demonstrated to undergo the noncanonical repeat-associated non-AUG translation (RAN translation) into pathologic dipeptide repeats in patient brains, a process that is thought to depend upon RNA structure. We previously demonstrated that the r(GGGGCC)n RNA forms repeat tract length-dependent G-quadruplex structures that bind the ASF/SF2 protein. Here we show that the cationic porphyrin (5,10,15,20-tetra(N-methyl-4-pyridyl) porphyrin (TMPyP4)), which can bind some G-quadruplex-forming sequences, can bind and distort the G-quadruplex formed by r(GGGGCC)8, and this ablates the interaction of either hnRNPA1 or ASF/SF2 with the repeat. These findings provide proof of concept that nucleic acid binding small molecules, such as TMPyP4, can distort the secondary structure of the C9orf72 repeat, which may beneficially disrupt protein interactions, which may ablate either protein sequestration and/or RAN translation into potentially toxic dipeptides. Disruption of secondary structure formation of the C9orf72 RNA repeats may be a viable therapeutic avenue, as well as a means to test the role of RNA structure upon RAN translation.

Reduction of erosion by protein-containing toothpastes.

PubMed

Jager, D H J; Vissink, A; Timmer, C J; Bronkhorst, E; Vieira, A M; Huysmans, M C D N J M

2013-01-01

To assess the effect of protein-containing toothpastes on the progression of dental erosion in situ (with pellicle) and in vitro (without pellicle). A combined split-mouth (extraoral water or toothpaste brushing) and crossover (type of toothpaste) setup was used. Two protein-containing (high/low concentrations of colostrum) and one nonprotein (placebo) toothpaste were investigated. Sixteen volunteers wore intraoral appliances containing 2 human enamel samples on 3 afternoons for pellicle growth during 90 min. One enamel sample was brushed for 5 s with one of the three toothpastes and subsequently exposed to a slurry of the corresponding toothpaste for 2 min. The other sample was exposed to water. Both samples were subsequently exposed to citric acid (extraorally). Loss of calcium and inorganic phosphate were determined. The same sequence of exposures was applied to 16 enamel samples in an in vitro setup without pellicle. With the in situ-formed pellicle, all toothpastes significantly reduced calcium loss compared to water brushing, although no significant differences were found among toothpastes (p = 0.073). For the loss of phosphate, a significant reduction could be found with the use of the high-protein toothpaste compared to the nonprotein toothpaste. Overall there were only slight differences between the toothpastes. Toothpaste effects were less clear in the in vitro experiment. The addition of proteins to toothpaste shows some promise for the prevention of erosion. Further research is needed to investigate the performance of the protein-containing toothpastes in longer in situ studies with regard to erosive wear. Copyright © 2012 S. Karger AG, Basel.

Fine mapping of Restorer-of-fertility in pepper (Capsicum annuum L.) identified a candidate gene encoding a pentatricopeptide repeat (PPR)-containing protein.

PubMed

Jo, Yeong Deuk; Ha, Yeaseong; Lee, Joung-Ho; Park, Minkyu; Bergsma, Alex C; Choi, Hong-Il; Goritschnig, Sandra; Kloosterman, Bjorn; van Dijk, Peter J; Choi, Doil; Kang, Byoung-Cheorl

2016-10-01

Using fine mapping techniques, the genomic region co-segregating with Restorer - of - fertility ( Rf ) in pepper was delimited to a region of 821 kb in length. A PPR gene in this region, CaPPR6 , was identified as a strong candidate for Rf based on expression pattern and characteristics of encoding sequence. Cytoplasmic-genic male sterility (CGMS) has been used for the efficient production of hybrid seeds in peppers (Capsicum annuum L.). Although the mitochondrial candidate genes that might be responsible for cytoplasmic male sterility (CMS) have been identified, the nuclear Restorer-of-fertility (Rf) gene has not been isolated. To identify the genomic region co-segregating with Rf in pepper, we performed fine mapping using an Rf-segregating population consisting of 1068 F2 individuals, based on BSA-AFLP and a comparative mapping approach. Through six cycles of chromosome walking, the co-segregating region harboring the Rf locus was delimited to be within 821 kb of sequence. Prediction of expressed genes in this region based on transcription analysis revealed four candidate genes. Among these, CaPPR6 encodes a pentatricopeptide repeat (PPR) protein with PPR motifs that are repeated 14 times. Characterization of the CaPPR6 protein sequence, based on alignment with other homologs, showed that CaPPR6 is a typical Rf-like (RFL) gene reported to have undergone diversifying selection during evolution. A marker developed from a sequence near CaPPR6 showed a higher prediction rate of the Rf phenotype than those of previously developed markers when applied to a panel of breeding lines of diverse origin. These results suggest that CaPPR6 is a strong candidate for the Rf gene in pepper.

An Unusual Hydrophobic Core Confers Extreme Flexibility to HEAT Repeat Proteins

PubMed Central

Kappel, Christian; Zachariae, Ulrich; Dölker, Nicole; Grubmüller, Helmut

2010-01-01

Alpha-solenoid proteins are suggested to constitute highly flexible macromolecules, whose structural variability and large surface area is instrumental in many important protein-protein binding processes. By equilibrium and nonequilibrium molecular dynamics simulations, we show that importin-β, an archetypical α-solenoid, displays unprecedentedly large and fully reversible elasticity. Our stretching molecular dynamics simulations reveal full elasticity over up to twofold end-to-end extensions compared to its bound state. Despite the absence of any long-range intramolecular contacts, the protein can return to its equilibrium structure to within 3 Å backbone RMSD after the release of mechanical stress. We find that this extreme degree of flexibility is based on an unusually flexible hydrophobic core that differs substantially from that of structurally similar but more rigid globular proteins. In that respect, the core of importin-β resembles molten globules. The elastic behavior is dominated by nonpolar interactions between HEAT repeats, combined with conformational entropic effects. Our results suggest that α-solenoid structures such as importin-β may bridge the molecular gap between completely structured and intrinsically disordered proteins. PMID:20816072

A novel Amoeba proteus 120 kDa actin-binding protein with only 1 filamin repeat and a coiled-coil region.

PubMed

Sobczak, Magdalena; Kocik, Elzbieta; Redowicz, Maria Jolanta

2007-02-01

A novel 120 kDa actin-binding protein (ApABP-F1) was found in Amoeba proteus. It was distributed throughout the cytoplasm, mainly in the subplasma membrane and perinuclear-nuclear areas, enriched in actin. The full-length cDNA of ApABP consisted of 2672 nucleotides with an open reading frame of 878 amino acids, giving a ~95 kDa protein with a theoretical pI value of 5.11. It had a novel domain organization pattern: the N terminus (residues 1-104) contained 1 calponin-homology (CH) domain, followed by only 1 region that was homologous to the filamin repeat (FR, residues 209-324), and a central region (residues 344-577) exhibiting a very high probability of coiled-coil formation, probably engaged in the observed protein dimerization. A phylogenetic tree constructed for CH domains from 25 various proteins revealed that the CH domain of ApABP was most related to that of the hypothetical mouse KIAA0903-like protein, whereas not much relationship to either filamins or the gelation factor (ABP-120) of Dictyostelium discoideum and Entamoeba histolytica was found.

Surface and bulk effects of K in Cu 1-xK xIn 1-yGa ySe 2 solar cells

DOE PAGES

Muzzillo, Christopher P.; Anderson, Timothy J.

2017-12-29

Two strategies for enhancing photovoltaic (PV) performance in chalcopyrite solar cells were investigated: Cu 1-xK xIn 1-yGa ySe 2 absorbers with low K content (K/(K+Cu), or x ~ 0.07) distributed throughout the bulk, and CuIn 1-yGa ySe 2 absorbers with KIn 1-yGa ySe 2 grown on their surfaces. Distributing K throughout the bulk absorbers improved power conversion efficiency, open-circuit voltage (VOC) and fill factor (FF) for Ga/(Ga+In) of 0, 0.3 and 0.5. Surface KIn 1-yGa ySe 2 and bulk x ~ 0.07 Cu 1-xK xIn 1-yGa ySe 2 films with Ga/(Ga+In), or y of 0.3 and 0.5 also had improvedmore » efficiency, VOC, and FF, relative to CuIn 1-yGa ySe 2 baselines. On the other hand, y ~ 1 absorbers did not benefit from K introduction. Similar to Cu 1-xK xInSe 2, the formation of Cu 1-xK xGaSe 2 alloys was favored at low temperatures and high Na supply by the substrate, relative to the formation of mixed-phase CuGaSe 2 + KGaSe 2. KIn 1-yGa ySe 2 alloys were grown for the first time, as evidenced by X-ray diffraction and ultraviolet/visible spectroscopy. For all Ga/(Ga+In) compositions, the surface KIn 1-yGa ySe 2 absorbers had superior PV performance in buffered and buffer-free devices. However, the bulk x ~ 0.07 absorbers only outperformed the baselines in buffered devices. The data demonstrate that KIn 1-yGa ySe 2 passivates the surface of CuIn 1-yGa ySe 2 to increase efficiency, VOC, and FF, while bulk Cu 1-xK xIn 1-yGa ySe 2 absorbers with x ~ 0.07 enhance efficiency, VOC, and FF by some other mechanism.« less

Surface and bulk effects of K in Cu 1-xK xIn 1-yGa ySe 2 solar cells

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

Muzzillo, Christopher P.; Anderson, Timothy J.

Two strategies for enhancing photovoltaic (PV) performance in chalcopyrite solar cells were investigated: Cu 1-xK xIn 1-yGa ySe 2 absorbers with low K content (K/(K+Cu), or x ~ 0.07) distributed throughout the bulk, and CuIn 1-yGa ySe 2 absorbers with KIn 1-yGa ySe 2 grown on their surfaces. Distributing K throughout the bulk absorbers improved power conversion efficiency, open-circuit voltage (VOC) and fill factor (FF) for Ga/(Ga+In) of 0, 0.3 and 0.5. Surface KIn 1-yGa ySe 2 and bulk x ~ 0.07 Cu 1-xK xIn 1-yGa ySe 2 films with Ga/(Ga+In), or y of 0.3 and 0.5 also had improvedmore » efficiency, VOC, and FF, relative to CuIn 1-yGa ySe 2 baselines. On the other hand, y ~ 1 absorbers did not benefit from K introduction. Similar to Cu 1-xK xInSe 2, the formation of Cu 1-xK xGaSe 2 alloys was favored at low temperatures and high Na supply by the substrate, relative to the formation of mixed-phase CuGaSe 2 + KGaSe 2. KIn 1-yGa ySe 2 alloys were grown for the first time, as evidenced by X-ray diffraction and ultraviolet/visible spectroscopy. For all Ga/(Ga+In) compositions, the surface KIn 1-yGa ySe 2 absorbers had superior PV performance in buffered and buffer-free devices. However, the bulk x ~ 0.07 absorbers only outperformed the baselines in buffered devices. The data demonstrate that KIn 1-yGa ySe 2 passivates the surface of CuIn 1-yGa ySe 2 to increase efficiency, VOC, and FF, while bulk Cu 1-xK xIn 1-yGa ySe 2 absorbers with x ~ 0.07 enhance efficiency, VOC, and FF by some other mechanism.« less

Cathepsins L and Z Are Critical in Degrading Polyglutamine-containing Proteins within Lysosomes*

PubMed Central

Bhutani, Nidhi; Piccirillo, Rosanna; Hourez, Raphael; Venkatraman, Prasanna; Goldberg, Alfred L.

2012-01-01

In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins. PMID:22451661

Hexanucleotide Repeats in ALS/FTD Form Length-Dependent RNA Foci, Sequester RNA Binding Proteins, and Are Neurotoxic

PubMed Central

Lee, Youn-Bok; Chen, Han-Jou; Peres, João N.; Gomez-Deza, Jorge; Attig, Jan; Štalekar, 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.

2013-01-01

Summary 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. PMID:24290757

History, rare, and multiple events of mechanical unfolding of repeat proteins

NASA Astrophysics Data System (ADS)

Sumbul, Fidan; Marchesi, Arin; Rico, Felix

2018-03-01

Mechanical unfolding of proteins consisting of repeat domains is an excellent tool to obtain large statistics. Force spectroscopy experiments using atomic force microscopy on proteins presenting multiple domains have revealed that unfolding forces depend on the number of folded domains (history) and have reported intermediate states and rare events. However, the common use of unspecific attachment approaches to pull the protein of interest holds important limitations to study unfolding history and may lead to discarding rare and multiple probing events due to the presence of unspecific adhesion and uncertainty on the pulling site. Site-specific methods that have recently emerged minimize this uncertainty and would be excellent tools to probe unfolding history and rare events. However, detailed characterization of these approaches is required to identify their advantages and limitations. Here, we characterize a site-specific binding approach based on the ultrastable complex dockerin/cohesin III revealing its advantages and limitations to assess the unfolding history and to investigate rare and multiple events during the unfolding of repeated domains. We show that this approach is more robust, reproducible, and provides larger statistics than conventional unspecific methods. We show that the method is optimal to reveal the history of unfolding from the very first domain and to detect rare events, while being more limited to assess intermediate states. Finally, we quantify the forces required to unfold two molecules pulled in parallel, difficult when using unspecific approaches. The proposed method represents a step forward toward more reproducible measurements to probe protein unfolding history and opens the door to systematic probing of rare and multiple molecule unfolding mechanisms.

Specific Binding of Tetratricopeptide Repeat Proteins to Heat Shock Protein 70 (Hsp70) and Heat Shock Protein 90 (Hsp90) Is Regulated by Affinity and Phosphorylation.

PubMed

Assimon, Victoria A; Southworth, Daniel R; Gestwicki, Jason E

2015-12-08

Heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) require the help of tetratricopeptide repeat (TPR) domain-containing cochaperones for many of their functions. Each monomer of Hsp70 or Hsp90 can interact with only a single TPR cochaperone at a time, and each member of the TPR cochaperone family brings distinct functions to the complex. Thus, competition for TPR binding sites on Hsp70 and Hsp90 appears to shape chaperone activity. Recent structural and biophysical efforts have improved our understanding of chaperone-TPR contacts, focusing on the C-terminal EEVD motif that is present in both chaperones. To better understand these important protein-protein interactions on a wider scale, we measured the affinity of five TPR cochaperones, CHIP, Hop, DnaJC7, FKBP51, and FKBP52, for the C-termini of four members of the chaperone family, Hsc70, Hsp72, Hsp90α, and Hsp90β, in vitro. These studies identified some surprising selectivity among the chaperone-TPR pairs, including the selective binding of FKBP51/52 to Hsp90α/β. These results also revealed that other TPR cochaperones are only able to weakly discriminate between the chaperones or between their paralogs. We also explored whether mimicking phosphorylation of serine and threonine residues near the EEVD motif might impact affinity and found that pseudophosphorylation had selective effects on binding to CHIP but not other cochaperones. Together, these findings suggest that both intrinsic affinity and post-translational modifications tune the interactions between the Hsp70 and Hsp90 proteins and the TPR cochaperones.

The solution structure of the pentatricopeptide repeat protein PPR10 upon binding atpH RNA

PubMed Central

Gully, Benjamin S.; Cowieson, Nathan; Stanley, Will A.; Shearston, Kate; Small, Ian D.; Barkan, Alice; Bond, Charles S.

2015-01-01

The pentatricopeptide repeat (PPR) protein family is a large family of RNA-binding proteins that is characterized by tandem arrays of a degenerate 35-amino-acid motif which form an α-solenoid structure. PPR proteins influence the editing, splicing, translation and stability of specific RNAs in mitochondria and chloroplasts. Zea mays PPR10 is amongst the best studied PPR proteins, where sequence-specific binding to two RNA transcripts, atpH and psaJ, has been demonstrated to follow a recognition code where the identity of two amino acids per repeat determines the base-specificity. A recently solved ZmPPR10:psaJ complex crystal structure suggested a homodimeric complex with considerably fewer sequence-specific protein–RNA contacts than inferred previously. Here we describe the solution structure of the ZmPPR10:atpH complex using size-exclusion chromatography-coupled synchrotron small-angle X-ray scattering (SEC-SY-SAXS). Our results support prior evidence that PPR10 binds RNA as a monomer, and that it does so in a manner that is commensurate with a canonical and predictable RNA-binding mode across much of the RNA–protein interface. PMID:25609698

The PDZ and band 4.1 containing protein Frmpd1 regulates the subcellular location of activator of G-protein signaling 3 and its interaction with G-proteins.

PubMed

An, Ningfei; Blumer, Joe B; Bernard, Michael L; Lanier, Stephen M

2008-09-05

Activator of G-protein signaling 3 (AGS3) is one of nine mammalian proteins containing one or more G-protein regulatory (GPR) motifs that stabilize the GDP-bound conformation of Galphai. Such proteins have revealed unexpected functional diversity for the "G-switch" in the control of events within the cell independent of the role of heterotrimeric G-proteins as transducers for G-protein-coupled receptors at the cell surface. A key question regarding this class of proteins is what controls their subcellular positioning and interaction with G-proteins. We conducted a series of yeast two-hybrid screens to identify proteins interacting with the tetratricopeptide repeat (TPR) of AGS3, which plays an important role in subcellular positioning of the protein. We report the identification of Frmpd1 (FERM and PDZ domain containing 1) as a regulatory binding partner of AGS3. Frmpd1 binds to the TPR domain of AGS3 and coimmunoprecipitates with AGS3 from cell lysates. Cell fractionation indicated that Frmpd1 stabilizes AGS3 in a membrane fraction. Upon cotransfection of COS7 cells with Frmpd1-GFP and AGS3-mRFP, AGS3-mRFP is observed in regions of the cell cortex and also in membrane extensions or processes where it appears to be colocalized with Frmpd1-GFP based upon the merged fluorescent signals. Frmpd1 knockdown (siRNA) in Cath.a-differentiated neuronal cells decreased the level of endogenous AGS3 in membrane fractions by approximately 50% and enhanced the alpha2-adrenergic receptor-mediated inhibition of forskolin-induced increases in cAMP. The coimmunoprecipitation of Frmpd1 with AGS3 is lost as the amount of Galphai3 in the cell is increased and AGS3 apparently switches its binding partner from Frmpd1 to Galphai3 indicating that the interaction of AGS3 with Frmpd1 and Galphai3 is mutually exclusive. Mechanistically, Frmpd1 may position AGS3 in a membrane environment where it then interacts with Galphai in a regulated manner.

Top surface blade residues and the central channel water molecules are conserved in every repeat of the integrin-like β-propeller structures.

PubMed

Denesyuk, Alexander; Denessiouk, Konstantin; Johnson, Mark S

2018-02-01

An integrin-like β-propeller domain contains seven repeats of a four-stranded antiparallel β-sheet motif (blades). Previously we described a 3D structural motif within each blade of the integrin-type β-propeller. Here, we show unique structural links that join different blades of the β-propeller structure, which together with the structural motif for a single blade are repeated in a β-propeller to provide the functional top face of the barrel, found to be involved in protein-protein interactions and substrate recognition. We compare functional top face diagrams of the integrin-type β-propeller domain and two non-integrin type β-propeller domains of virginiamycin B lyase and WD Repeat-Containing Protein 5. Copyright © 2017 Elsevier Inc. All rights reserved.

Group B Streptococcal Serine-Rich Repeat Proteins Promote Interaction With Fibrinogen and Vaginal Colonization

PubMed Central

Wang, Nai-Yu; Patras, Kathryn A.; Seo, Ho Seong; Cavaco, Courtney K.; Rösler, Berenice; Neely, Melody N.; Sullam, Paul M.; Doran, Kelly S.

2014-01-01

Group B streptococcus (GBS) can cause severe disease in susceptible hosts, including newborns, pregnant women, and the elderly. GBS serine-rich repeat (Srr) surface glycoproteins are important adhesins/invasins in multiple host tissues, including the vagina. However, exact molecular mechanisms contributing to their importance in colonization are unknown. We have recently determined that Srr proteins contain a fibrinogen-binding region (BR) and hypothesize that Srr-mediated fibrinogen binding may contribute to GBS cervicovaginal colonization. In this study, we observed that fibrinogen enhanced wild-type GBS attachment to cervical and vaginal epithelium, and that this was dependent on Srr1. Moreover, purified Srr1-BR peptide bound directly to host cells, and peptide administration in vivo reduced GBS recovery from the vaginal tract. Furthermore, a GBS mutant strain lacking only the Srr1 “latching” domain exhibited decreased adherence in vitro and decreased persistence in a mouse model of GBS vaginal colonization, suggesting the importance of Srr–fibrinogen interactions in the female reproductive tract. PMID:24620021

Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

NASA Technical Reports Server (NTRS)

Carter, Daniel C. (Inventor)

1998-01-01

In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

Poly-dipeptides encoded by the C9ORF72 repeats block global protein translation.

PubMed

Kanekura, Kohsuke; Yagi, Takuya; Cammack, Alexander J; Mahadevan, Jana; Kuroda, Masahiko; Harms, Matthew B; Miller, Timothy M; Urano, Fumihiko

2016-05-01

The expansion of the GGGGCC hexanucleotide repeat in the non-coding region of the Chromosome 9 open-reading frame 72 (C9orf72) gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). This genetic alteration leads to the accumulation of five types of poly-dipeptides translated from the GGGGCC hexanucleotide repeat. Among these, poly-proline-arginine (poly-PR) and poly-glycine-arginine (poly-GR) peptides are known to be neurotoxic. However, the mechanisms of neurotoxicity associated with these poly-dipeptides are not clear. A proteomics approach identified a number of interacting proteins with poly-PR peptide, including mRNA-binding proteins, ribosomal proteins, translation initiation factors and translation elongation factors. Immunostaining of brain sections from patients with C9orf72 ALS showed that poly-GR was colocalized with a mRNA-binding protein, hnRNPA1. In vitro translation assays showed that poly-PR and poly-GR peptides made insoluble complexes with mRNA, restrained the access of translation factors to mRNA, and blocked protein translation. Our results demonstrate that impaired protein translation mediated by poly-PR and poly-GR peptides plays a role in neurotoxicity and reveal that the pathways altered by the poly-dipeptides-mRNA complexes are potential therapeutic targets for treatment of C9orf72 FTD/ALS. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

PubMed

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

2016-05-24

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.

A screen of cell-surface molecules identifies leucine-rich repeat proteins as key mediators of synaptic target selection in the Drosophila neuromuscular system

PubMed Central

Kurusu, Mitsuhiko; Cording, Amy; Taniguchi, Misako; Menon, Kaushiki; Suzuki, Emiko; Zinn, Kai

2008-01-01

Summary In Drosophila embryos and larvae, a small number of identified motor neurons innervate body wall muscles in a highly stereotyped pattern. Although genetic screens have identified many proteins that are required for axon guidance and synaptogenesis in this system, little is known about the mechanisms by which muscle fibers are defined as targets for specific motor axons. To identify potential target labels, we screened 410 genes encoding cell-surface and secreted proteins, searching for those whose overexpression on all muscle fibers causes motor axons to make targeting errors. Thirty such genes were identified, and a number of these were members of a large gene family encoding proteins whose extracellular domains contain leucine-rich repeat (LRR) sequences, which are protein interaction modules. By manipulating gene expression in muscle 12, we showed that four LRR proteins participate in the selection of this muscle as the appropriate synaptic target for the RP5 motor neuron. PMID:18817735

Classification of proteins with shared motifs and internal repeats in the ECOD database

PubMed Central

Kinch, Lisa N.; Liao, Yuxing

2016-01-01

Abstract Proteins and their domains evolve by a set of events commonly including the duplication and divergence of small motifs. The presence of short repetitive regions in domains has generally constituted a difficult case for structural domain classifications and their hierarchies. We developed the Evolutionary Classification Of protein Domains (ECOD) in part to implement a new schema for the classification of these types of proteins. Here we document the ways in which ECOD classifies proteins with small internal repeats, widespread functional motifs, and assemblies of small domain‐like fragments in its evolutionary schema. We illustrate the ways in which the structural genomics project impacted the classification and characterization of new structural domains and sequence families over the decade. PMID:26833690

DNA triplet repeats mediate heterochromatin-protein-1-sensitive variegated gene silencing.

PubMed

Saveliev, Alexander; Everett, Christopher; Sharpe, Tammy; Webster, Zoë; Festenstein, Richard

2003-04-24

Gene repression is crucial to the maintenance of differentiated cell types in multicellular organisms, whereas aberrant silencing can lead to disease. The organization of DNA into chromatin and heterochromatin is implicated in gene silencing. In chromatin, DNA wraps around histones, creating nucleosomes. Further condensation of chromatin, associated with large blocks of repetitive DNA sequences, is known as heterochromatin. Position effect variegation (PEV) occurs when a gene is located abnormally close to heterochromatin, silencing the affected gene in a proportion of cells. Here we show that the relatively short triplet-repeat expansions found in myotonic dystrophy and Friedreich's ataxia confer variegation of expression on a linked transgene in mice. Silencing was correlated with a decrease in promoter accessibility and was enhanced by the classical PEV modifier heterochromatin protein 1 (HP1). Notably, triplet-repeat-associated variegation was not restricted to classical heterochromatic regions but occurred irrespective of chromosomal location. Because the phenomenon described here shares important features with PEV, the mechanisms underlying heterochromatin-mediated silencing might have a role in gene regulation at many sites throughout the mammalian genome and modulate the extent of gene silencing and hence severity in several triplet-repeat diseases.

Entry into the nuclear pore complex is controlled by a cytoplasmic exclusion zone containing dynamic GLFG-repeat nucleoporin domains.

PubMed

Fiserova, Jindriska; Spink, Matthew; Richards, Shane A; Saunter, Christopher; Goldberg, Martin W

2014-01-01

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic movement. The central channel contains proteins with phenylalanine-glycine (FG) repeats, or variations (GLFG, glycine-leucine-phenylalanine-glycine). These are 'intrinsically disordered' and often represent weak interaction sites that become ordered upon interaction. We investigated this possibility during nuclear transport. Using electron microscopy of S. cerevisiae, we show that NPC cytoplasmic filaments form a dome-shaped structure enclosing GLFG domains. GLFG domains extend out of this structure and are part of an 'exclusion zone' that might act as a partial barrier to entry of transport-inert proteins. The anchor domain of a GLFG nucleoporin locates exclusively to the central channel. By contrast, the localisation of the GLFG domains varied between NPCs and could be cytoplasmic, central or nucleoplasmic and could stretch up to 80 nm. These results suggest a dynamic exchange between ordered and disordered states. In contrast to diffusion through the NPC, transport cargoes passed through the exclusion zone and accumulated near the central plane. We also show that movement of cargo through the NPC is accompanied by relocation of GLFG domains, suggesting that binding, restructuring and movement of these domains could be part of the translocation mechanism.

The protein network surrounding the human telomere repeat binding factors TRF1, TRF2, and POT1

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

Giannone, Richard J; McDonald, W Hayes; Hurst, Gregory

Telomere integrity (including telomere length and capping) is critical in overall genomic stability. Telomere repeat binding factors and their associated proteins play vital roles in telomere length regulation and end protection. In this study, we explore the protein network surrounding telomere repeat binding factors, TRF1, TRF2, and POT1 using dual-tag affinity purification in combination with multidimensional protein identification technology liquid chromatography - tandem mass spectrometry (MudPIT LC-MS/MS). After control subtraction and data filtering, we found that TRF2 and POT1 co-purified all six members of the telomere protein complex, while TRF1 identified five of six components at frequencies that lend evidencemore » towards the currently accepted telomere architecture. Many of the known TRF1 or TRF2 interacting proteins were also identified. Moreover, putative associating partners identified for each of the three core components fell into functional categories such as DNA damage repair, ubiquitination, chromosome cohesion, chromatin modification/remodeling, DNA replication, cell cycle and transcription regulation, nucleotide metabolism, RNA processing, and nuclear transport. These putative protein-protein associations may participate in different biological processes at telomeres or, intriguingly, outside telomeres.« less

Liking of health-functional foods containing lupin kernel fibre following repeated consumption in a dietary intervention setting.

PubMed

Hall, Ramon S; Baxter, Amynta L; Fryirs, Cathy; Johnson, Stuart K

2010-10-01

Liking of a particular food after repeated consumption may be reduced, limiting the effectiveness of health-functional foods requiring on-going consumption to deliver their benefits. This study examined the effect of repeated consumption of foods containing the novel ingredient, Australian sweet lupin (Lupinus angustifolius) kernel fibre (LKFibre) on sensory acceptability in the dietary intervention setting. In a single-blind randomised crossover 4-week intervention, participants consumed both control and equivalent LKFibre-containing products daily on separate interventions separated by a 4-week period on habitual diet. Seven products: muesli, bread, muffin, chocolate brownie, chocolate milk drink, pasta and instant mashed potato were assessed twice (days 4 and 18 of intervention), by 38 participants for appearance, texture, flavour and general acceptability using a structured graphic hedonic scale. Overall the results showed there was no reduction (P=0.594) in general acceptability of LKFibre foods after repeated consumption, suggesting potential for long-term consumption. The control food products were however generally preferred (P<0.001) over the LKFibre foods; the mean difference for general acceptability between being <6% (0.82cm) of the 15cm hedonic scale used, suggesting LKF addition did not severely affect product palatability.

Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation

PubMed Central

Casillas-Ituarte, Nadia N.; Cruz, Carlos H. B.; Lins, Roberto D.; DiBartola, Alex C.; Howard, Jessica; Liang, Xiaowen; Höök, Magnus; Viana, Isabelle F. T.; Sierra-Hernández, M. Roxana; Lower, Steven K.

2017-01-01

The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands (e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/koff) and dissociation constants (Kd = koff/kon), determined by mechanically dissociating the Fn·peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 μm) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 μm). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I (Kdapp = 0.2–0.5 μm, as determined by SPR) compared with the lowest-affinity double-alanine peptide (Kdapp = 3.8 μm). Together, these findings demonstrate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections. PMID:28400484

Heat shock protein-containing exosomes in mid-trimester amniotic fluids.

PubMed

Asea, Alexzander; Jean-Pierre, Claudel; Kaur, Punit; Rao, Preethi; Linhares, Iara M; Skupski, Daniel; Witkin, Steven S

2008-10-01

Exosomes are multivesicular bodies formed by inverse membrane budding into the lumen of an endocytic compartment. Fusion with the plasma membrane leads to their release into the external milieu. The incorporation of heat shock proteins into exosomes has been associated with immune regulatory activity. We have examined whether heat shock protein-containing exosomes are present in mid-trimester amniotic fluid. Exosomes were isolated from mid-trimester amniotic fluids by sequential low-speed and high-speed centrifugation followed by sucrose density gradient centrifugation. Biochemical characterization included floatation pattern in sucrose gradients, acetylcholinesterase (AChE) activity and Western blot analysis for exosome-containing proteins. Exosomes were present in each of 23 amniotic fluids tested. They banded at a density of 1.17g/ml in sucrose gradients, were positive for AChE activity and contained tubulin, the inducible 72kDa heat shock protein, Hsp72 and the constitutively expressed heat shock protein, Hsc73; they were negative for calnexin. Exosome concentrations correlated positively with the number of pregnancies. Heat shock protein-containing exosomes are constituents of mid-trimester amniotic fluids and may contribute to immune regulation within the amniotic cavity.

Membrane cofactor protein (CD46) is a keratinocyte receptor for the M protein of the group A streptococcus.

PubMed

Okada, N; Liszewski, M K; Atkinson, J P; Caparon, M

1995-03-28

The pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is the causative agent of numerous suppurative diseases of human skin. The M protein of S. pyogenes mediates the adherence of the bacterium to keratinocytes, the most numerous cell type in the epidermis. In this study, we have constructed and analyzed a series of mutant M proteins and have shown that the C repeat domain of the M molecule is responsible for cell recognition. The binding of factor H, a serum regulator of complement activation, to the C repeat region of M protein blocked bacterial adherence. Factor H is a member of a large family of complement regulatory proteins that share a homologous structural motif termed the short consensus repeat. Membrane cofactor protein (MCP), or CD46, is a short consensus repeat-containing protein found on the surface of keratinocytes, and purified MCP could competitively inhibit the adherence of S. pyogenes to these cells. Furthermore, the M protein was found to bind directly to MCP, whereas mutant M proteins that lacked the C repeat domain did not bind MCP, suggesting that recognition of MCP plays an important role in the ability of the streptococcus to adhere to keratinocytes.

The Molecular Chaperone TRiC/CCT Binds to the Trp-Asp 40 (WD40) Repeat Protein WDR68 and Promotes Its Folding, Protein Kinase DYRK1A Binding, and Nuclear Accumulation*

PubMed Central

Miyata, Yoshihiko; Shibata, Takeshi; Aoshima, Masato; Tsubata, Takuichi; Nishida, Eisuke

2014-01-01

Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68. PMID

A Variable Polyglutamine Repeat Affects Subcellular Localization and Regulatory Activity of a Populus ANGUSTIFOLIA Protein.

PubMed

Bryan, Anthony C; Zhang, Jin; Guo, Jianjun; Ranjan, Priya; Singan, Vasanth; Barry, Kerrie; Schmutz, Jeremy; Weighill, Deborah; Jacobson, Daniel; Jawdy, Sara; Tuskan, Gerald A; Chen, Jin-Gui; Muchero, Wellington

2018-06-08

Polyglutamine (polyQ) stretches have been reported to occur in proteins across many organisms including animals, fungi and plants. Expansion of these repeats has attracted much attention due their associations with numerous human diseases including Huntington's and other neurological maladies. This suggests that the relative length of polyQ stretches is an important modulator of their function. Here, we report the identification of a Populus C-terminus binding protein (CtBP) ANGUSTIFOLIA ( PtAN1 ) which contains a polyQ stretch whose functional relevance had not been established. Analysis of 917 resequenced Populus trichocarpa genotypes revealed three allelic variants at this locus encoding 11-, 13- and 15-glutamine residues. Transient expression assays using Populus leaf mesophyll protoplasts revealed that the 11Q variant exhibited strong nuclear localization whereas the 15Q variant was only found in the cytosol, with the 13Q variant exhibiting localization in both subcellular compartments. We assessed functional implications by evaluating expression changes of putative PtAN1 targets in response to overexpression of the three allelic variants and observed allele-specific differences in expression levels of putative targets. Our results provide evidence that variation in polyQ length modulates PtAN1 function by altering subcellular localization. Copyright © 2018, G3: Genes, Genomes, Genetics.

Sfr13, a member of a large family of asymmetrically localized Sfi1-repeat proteins, is important for basal body separation and stability in Tetrahymena thermophila

PubMed Central

Stemm-Wolf, Alexander J.; Meehl, Janet B.; Winey, Mark

2013-01-01

Summary Directed fluid flow, which is achieved by the coordinated beating of motile cilia, is required for processes as diverse as cellular swimming, developmental patterning and mucus clearance. Cilia are nucleated, anchored and aligned at the plasma membrane by basal bodies, which are cylindrical microtubule-based structures with ninefold radial symmetry. In the unicellular ciliate Tetrahymena thermophila, two centrin family members associated with the basal body are important for both basal body organization and stabilization. We have identified a family of 13 proteins in Tetrahymena that contain centrin-binding repeats related to those identified in the Saccharomyces cerevisiae Sfi1 protein. We have named these proteins Sfr1–Sfr13 (for Sfi1-repeat). Nine of the Sfr proteins localize in unique polarized patterns surrounding the basal body, suggesting non-identical roles in basal body organization and association with basal body accessory structures. Furthermore, the Sfr proteins are found in distinct basal body populations in Tetrahymena cells, indicating that they are responsive to particular developmental programs. A complete genetic deletion of one of the family members, Sfr13, causes unstable basal bodies and defects in daughter basal body separation from the mother, phenotypes also observed with centrin disruption. It is likely that the other Sfr family members are involved in distinct centrin functions, providing specificity to the tasks that centrins perform at basal bodies. PMID:23426847

Mitogen-activated protein kinase is required for the behavioral desensitization that occurs after repeated injections of angiotensin II

PubMed Central

Vento, Peter J.; Daniels, Derek

2013-01-01

Angiotensin II (AngII) acts on central angiotensin type 1 (AT1) receptors to increase water and saline intake. Prolonged exposure to AngII in cell culture models results in a desensitization of the AT1 receptor that is thought to involve receptor internalization, and a behavioral correlate of this desensitization has been shown in rats after repeated central injections of AngII. Specifically, rats given repeated injections of AngII drink less water than controls after a subsequent test injection of AngII. Under the same conditions, however, repeated injections of AngII have no effect on AngII-induced saline intake. Given earlier studies indicating that separate intracellular signaling pathways mediate AngII-induced water and saline intake, we hypothesized that the desensitization observed in rats may be incomplete, leaving the receptor able to activate mitogen-activated protein (MAP) kinases (ERK1/2), which play a role in AngII-induced saline intake without affecting water intake. In support of this hypothesis, we found no difference in MAP kinase phosphorylation after an AngII test injection in rats given prior treatment with repeated injections of vehicle, AngII, or Sar1,Ile4,Ile8-AngII (SII), an AngII analog that activates MAP kinase without G protein coupling. In addition, we found that pretreatment with the MAP kinase inhibitor U0126 completely blocked the desensitizing effect of repeated AngII injections on water intake. Furthermore, AngII-induced water intake was reduced similarly by repeated injections of AngII or SII. The results suggest that G protein-independent signaling is sufficient to produce behavioral desensitization of the angiotensin system and that the desensitization requires MAP kinase activation. PMID:22581747

Mitogen-activated protein kinase is required for the behavioural desensitization that occurs after repeated injections of angiotensin II.

PubMed

Vento, Peter J; Daniels, Derek

2012-12-01

Angiotensin II (Ang II) acts on central angiotensin type 1 (AT(1)) receptors to increase water and saline intake. Prolonged exposure to Ang II in cell culture models results in a desensitization of the AT(1) receptor that is thought to involve receptor internalization, and a behavioural correlate of this desensitization has been shown in rats after repeated central injections of Ang II. Specifically, rats given repeated injections of Ang II drink less water than control animals after a subsequent test injection of Ang II. In the same conditions, however, repeated injections of Ang II have no effect on Ang II-induced saline intake. Given earlier studies indicating that separate intracellular signalling pathways mediate Ang II-induced water and saline intake, we hypothesized that the desensitization observed in rats may be incomplete, leaving the receptor able to activate mitogen-activated protein (MAP) kinases (ERK1/2), which play a role in Ang II-induced saline intake without affecting water intake. In support of this hypothesis, we found no difference in MAP kinase phosphorylation after an Ang II test injection in rats given prior treatment with repeated injections of vehicle, Ang II or Sar(1),Ile(4),Ile(8)-Ang II (SII), an Ang II analogue that activates MAP kinase without G protein coupling. In addition, we found that pretreatment with the MAP kinase inhibitor U0126 completely blocked the desensitizing effect of repeated Ang II injections on water intake. Furthermore, Ang II-induced water intake was reduced to a similar extent by repeated injections of Ang II or SII. The results suggest that G protein-independent signalling is sufficient to produce behavioural desensitization of the angiotensin system and that the desensitization requires MAP kinase activation.

Evolutionary dynamics of the immunodominant repeats of the Plasmodium vivax malaria-vaccine candidate circumsporozoite protein (CSP)

PubMed Central

Patil, Aarti; Orjuela-Sánchez, Pamela; da Silva-Nunes, Mônica; Ferreira, Marcelo U.

2010-01-01

The circumsporozoite protein (CSP) of Plasmodium vivax, a major target for malaria vaccine development, has immunodominant B-cell epitopes mapped to central nonapeptide repeat arrays. To determine whether rearrangements of repeat motifs during mitotic DNA replication of parasites create significant CSP diversity under conditions of low effective meiotic recombination rates, we examined csp alleles from sympatric P. vivax isolates systematically sampled from an area of low malaria endemicity in Brazil over a period of 14 months. Nine unique csp types, comprising six different nonapeptide repeats, were observed in 45 isolates analyzed. Identical or nearly identical repeats predominated in most arrays, consistent with their recent expansion. We found strong linkage disequilibrium at sites across the chromosome 8 segment flanking the csp locus, consistent with rare meiotic recombination in this region. We conclude that CSP repeat diversity may not be severely constrained by rare meiotic recombination in areas of low malaria endemicity. New repeat variants may be readily created by nonhomologous recombination even when meiotic recombination is rare, with potential implications for CSP-based vaccine development. PMID:20097310

Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

PubMed Central

Jazurek, Magdalena; Ciesiolka, Adam; Starega-Roslan, Julia; Bilinska, Katarzyna; Krzyzosiak, Wlodzimierz J.

2016-01-01

RNA–protein complexes play a central role in the regulation of fundamental cellular processes, such as mRNA splicing, localization, translation and degradation. The misregulation of these interactions can cause a variety of human diseases, including cancer and neurodegenerative disorders. Recently, many strategies have been developed to comprehensively analyze these complex and highly dynamic RNA–protein networks. Extensive efforts have been made to purify in vivo-assembled RNA–protein complexes. In this review, we focused on commonly used RNA-centric approaches that involve mass spectrometry, which are powerful tools for identifying proteins bound to a given RNA. We present various RNA capture strategies that primarily depend on whether the RNA of interest is modified. Moreover, we briefly discuss the advantages and limitations of in vitro and in vivo approaches. Furthermore, we describe recent advances in quantitative proteomics as well as the methods that are most commonly used to validate robust mass spectrometry data. Finally, we present approaches that have successfully identified expanded repeat-binding proteins, which present abnormal RNA–protein interactions that result in the development of many neurological diseases. PMID:27625393

Leucine-rich Repeats of Bacterial Surface Proteins Serve as Common Pattern Recognition Motifs of Human Scavenger Receptor gp340*

PubMed Central

Loimaranta, Vuokko; Hytönen, Jukka; Pulliainen, Arto T.; Sharma, Ashu; Tenovuo, Jorma; Strömberg, Nicklas; Finne, Jukka

2009-01-01

Scavenger receptors are innate immune molecules recognizing and inducing the clearance of non-host as well as modified host molecules. To recognize a wide pattern of invading microbes, many scavenger receptors bind to common pathogen-associated molecular patterns, such as lipopolysaccharides and lipoteichoic acids. Similarly, the gp340/DMBT1 protein, a member of the human scavenger receptor cysteine-rich protein family, displays a wide ligand repertoire. The peptide motif VEVLXXXXW derived from its scavenger receptor cysteine-rich domains is involved in some of these interactions, but most of the recognition mechanisms are unknown. In this study, we used mass spectrometry sequencing, gene inactivation, and recombinant proteins to identify Streptococcus pyogenes protein Spy0843 as a recognition receptor of gp340. Antibodies against Spy0843 are shown to protect against S. pyogenes infection, but no function or host receptor have been identified for the protein. Spy0843 belongs to the leucine-rich repeat (Lrr) family of eukaryotic and prokaryotic proteins. Experiments with truncated forms of the recombinant proteins confirmed that the Lrr region is needed in the binding of Spy0843 to gp340. The same motif of two other Lrr proteins, LrrG from the Gram-positive S. agalactiae and BspA from the Gram-negative Tannerella forsythia, also mediated binding to gp340. Moreover, inhibition of Spy0843 binding occurred with peptides containing the VEVLXXXXW motif, but also peptides devoid of the XXXXW motif inhibited binding of Lrr proteins. These results thus suggest that the conserved Lrr motif in bacterial proteins serves as a novel pattern recognition motif for unique core peptides of human scavenger receptor gp340. PMID:19465482

ROMP- and RAFT-Based Guanidinium-Containing Polymers as Scaffolds for Protein Mimic Synthesis.

PubMed

Sarapas, Joel M; Backlund, Coralie M; deRonde, Brittany M; Minter, Lisa M; Tew, Gregory N

2017-05-17

Cell-penetrating peptides are an important class of molecules with promising applications in bioactive cargo delivery. A diverse series of guanidinium-containing polymeric cell-penetrating peptide mimics (CPPMs) with varying backbone chemistries was synthesized and assessed for delivery of both GFP and fluorescently tagged siRNA. Specifically, we examined CPPMs based on norbornene, methacrylate, and styrene backbones to determine how backbone structure impacted internalization of these two cargoes. Either charge content or degree of polymerization was held constant at 20, with diguanidinium norbornene molecules being polymerized to both 10 and 20 repeat units. Generally, homopolymer CPPMs delivered low amounts of siRNA into Jurkat T cells, with no apparent backbone dependence; however, by adding a short hydrophobic methyl methacrylate block to the guanidinium-rich methacrylate polymer, siRNA delivery to nearly the entire cell population was achieved. Protein internalization yielded similar results for most of the CPPMs, though the block polymer was unable to deliver proteins. In contrast, the styrene-based CPPM yielded the highest internalization for GFP (≈40 % of cells affected), showing that indeed backbone chemistry impacts protein delivery, specifically through the incorporation of an aromatic group. These results demonstrate that an understanding of how polymer structure affects cargo-dependent internalization is critical to designing new, more effective CPPMs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Collagen Triple Helix Repeat Containing-1 (CTHRC1) Expression in Oral Squamous Cell Carcinoma (OSCC): Prognostic Value and Clinico-Pathological Implications

PubMed Central

Lee, Chia Ee; Vincent-Chong, Vui King; Ramanathan, Anand; Kallarakkal, Thomas George; Karen-Ng, Lee Peng; Ghani, Wan Maria Nabillah; Rahman, Zainal Ariff Abdul; Ismail, Siti Mazlipah; Abraham, Mannil Thomas; Tay, Keng Kiong; Mustafa, Wan Mahadzir Wan; Cheong, Sok Ching; Zain, Rosnah Binti

2015-01-01

BACKGROUND: Collagen Triple Helix Repeat Containing 1 (CTHRC1) is a protein often found to be over-expressed in various types of human cancers. However, correlation between CTHRC1 expression level with clinico-pathological characteristics and prognosis in oral cancer remains unclear. Therefore, this study aimed to determine mRNA and protein expression of CTHRC1 in oral squamous cell carcinoma (OSCC) and to evaluate the clinical and prognostic impact of CTHRC1 in OSCC. METHODS: In this study, mRNA and protein expression of CTHRC1 in OSCCs were determined by quantitative PCR and immunohistochemistry, respectively. The association between CTHRC1 and clinico-pathological parameters were evaluated by univariate and multivariate binary logistic regression analyses. Correlation between CTHRC1 protein expressions with survival were analysed using Kaplan-Meier and Cox regression models. RESULTS: Current study demonstrated CTHRC1 was significantly overexpressed at the mRNA level in OSCC. Univariate analyses indicated a high-expression of CTHRC1 that was significantly associated with advanced stage pTNM staging, tumour size ≥ 4 cm and positive lymph node metastasis (LNM). However, only positive LNM remained significant after adjusting with other confounder factors in multivariate logistic regression analyses. Kaplan-Meier survival analyses and Cox model demonstrated that patients with high-expression of CTHRC1 protein were associated with poor prognosis and is an independent prognostic factor in OSCC. CONCLUSION: This study indicated that over-expression of CTHRC1 potentially as an independent predictor for positive LNM and poor prognosis in OSCC. PMID:26664254

Inhibition of protein carbamylation in urea solution using ammonium-containing buffers.

PubMed

Sun, Shisheng; Zhou, Jian-Ying; Yang, Weiming; Zhang, Hui

2014-02-01

Urea solution is one of the most commonly employed protein denaturants for protease digestion in proteomic studies. However, it has long been recognized that urea solution can cause carbamylation at the N termini of proteins/peptides and at the side chain amino groups of lysine and arginine residues. Protein/peptide carbamylation blocks protease digestion and affects protein identification and quantification in mass spectrometry analysis by blocking peptide amino groups from isotopic/isobaric labeling and changing peptide charge states, retention times, and masses. In addition, protein carbamylation during sample preparation makes it difficult to study in vivo protein carbamylation. In this study, we compared the peptide carbamylation in urea solutions of different buffers and found that ammonium-containing buffers were the most effective buffers to inhibit protein carbamylation in urea solution. The possible mechanism of carbamylation inhibition by ammonium-containing buffers is discussed, and a revised procedure for the protease digestion of proteins in urea and ammonium-containing buffers was developed to facilitate its application in proteomic research. Copyright © 2013 Elsevier Inc. All rights reserved.

Inhibition of Protein Carbamylation in Urea Solution Using Ammonium Containing Buffers

PubMed Central

Sun, Shisheng; Zhou, Jian-Ying; Yang, Weiming; Zhang, Hui

2013-01-01

Urea solution is one of the most commonly employed protein denaturants for protease digestion in proteomic studies. However, it has long been recognized that urea solution can cause carbamylation at the N-termini of proteins/peptides and at the side chain amino groups of lysine and arginine residues. Protein/peptide carbamylation blocks protease digestion and affects protein identification and quantification in mass spectrometry analysis by blocking peptide amino groups from isotopic/isobaric labeling and changing peptide charge states, retention times and masses. In addition, protein carbamylation during sample preparation makes it difficult to study in vivo protein carbamylation. In this study, we compared the peptide carbamylation in urea solutions of different buffers and found that ammonium containing buffers were the most effective buffers to inhibit protein carbamylation in urea solution. The possible mechanism of carbamylation inhibition by ammonium containing buffers is discussed, and a revised procedure for the protease digestion of proteins in urea and ammonium containing buffers was developed to facilitate its application in proteomic research. PMID:24161613

MicroRNA-137 dysregulation predisposes to osteoporotic fracture by impeding ALP activity and expression via suppression of leucine-rich repeat-containing G-protein-coupled receptor 4 expression.

PubMed

Liu, Xiangjun; Xu, Xiaohui

2018-08-01

Osteoporosis is defined as a loss of bone mass and deterioration of its architecture resulting in bone weakness, which becomes prone to fracture. The objective of this study was to investigate the molecular mechanism by which miR-137 can reduce the risk of fracture in patients with osteoporosis. An online miRNA database and a luciferase reporter assay system were used to confirm that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) was the target of miR-137. Real-time PCR and western blot analysis were used to study miR-137 mRNA, the expression of LGR4 mRNA and protein among different groups or cells transfected with a scrambled miRNA control, miR-137 mimic, LGR4 siRNA and miR-137 inhibitor. Expression of miR-137 was upregulated to higher levels in cells isolated from osteoporosis patients with fracture than in those without fracture. The 'seed sequence' was found to be located within the 3' untranslated region (3'-UTR) of LGR4 mRNA by searching an online miRNA database. Luciferase reporter assay was performed to confirm that LGR4 is a direct target gene of miR-137 with a potential binding site in the 3'UTR of LGR4. Luciferase activity of cells transfected with wild-type LGR4 3'UTR was much lower than that of the cells transfected with mutant LGR4 3'UTR. The results of real-time PCR and immunohistochemistry experiments demonstrated that the expression levels of LGR4 mRNA and protein were much higher in osteoporosis patients with fracture than osteoporosis patients without fracture. We found that the expression levels of LGR4 mRNA and protein were clearly upregulated following transfection with miR-137 inhibitor, while noticeably downregulated following transfection with miR-137 mimic when compared with the scramble control. Furthermore, the expression of ALP mRNA and ALP activity in bone tissue were much higher in osteoporosis patients with fracture than those without fracture. In conclusion, these data prove that the overexpression of

Human telomeres that contain (CTAGGG)n repeats show replication dependent instability in somatic cells and the male germline

PubMed Central

Mendez-Bermudez, Aaron; Hills, Mark; Pickett, Hilda A.; Phan, Anh Tuân; Mergny, Jean-Louis; Riou, Jean-François; Royle, Nicola J.

2009-01-01

A number of different processes that impact on telomere length dynamics have been identified but factors that affect the turnover of repeats located proximally within the telomeric DNA are poorly defined. We have identified a particular repeat type (CTAGGG) that is associated with an extraordinarily high mutation rate (20% per gamete) in the male germline. The mutation rate is affected by the length and sequence homogeneity of the (CTAGGG)n array. This level of instability was not seen with other sequence-variant repeats, including the TCAGGG repeat type that has the same composition. Telomeres carrying a (CTAGGG)n array are also highly unstable in somatic cells with the mutation process resulting in small gains or losses of repeats that also occasionally result in the deletion of the whole (CTAGGG)n array. These sequences are prone to quadruplex formation in vitro but adopt a different topology from (TTAGGG)n (see accompanying article). Interestingly, short (CTAGGG)2 oligonucleotides induce a DNA damage response (γH2AX foci) as efficiently as (TTAGGG)2 oligos in normal fibroblast cells, suggesting they recruit POT1 from the telomere. Moreover, in vitro assays show that (CTAGGG)n repeats bind POT1 more efficiently than (TTAGGG)n or (TCAGGG)n. We estimate that 7% of human telomeres contain (CTAGGG)n repeats and when present, they create additional problems that probably arise during telomere replication. PMID:19656953

Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products

PubMed Central

Ludwig, Linda B; Ambrus, Julian L; Krawczyk, Kristie A; Sharma, Sanjay; Brooks, Stephen; Hsiao, Chiu-Bin; Schwartz, Stanley A

2006-01-01

Background While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ) protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). Results Inspection of published sequences revealed a potential transcription initiator element (INR) situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR) suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s) could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s) were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK) sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP) sequences identified HAPs from HIV+ human peripheral blood lymphocytes. Conclusion HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The novel HAPs are

Accumulation of sulfonamide resistance genes in arable soils due to repeated application of manure containing sulfadiazine.

PubMed

Heuer, Holger; Solehati, Qodiah; Zimmerling, Ute; Kleineidam, Kristina; Schloter, Michael; Müller, Tanja; Focks, Andreas; Thiele-Bruhn, Sören; Smalla, Kornelia

2011-04-01

Two soils were amended three times with pig manure. The abundance of sulfonamide resistance genes was determined by quantitative PCR 2 months after each application. In both soils treated with sulfadiazine-containing manure, the numbers of copies of sul1 and sul2 significantly increased compared to numbers after treatments with antibiotic-free manure or a control and accumulated with repeated applications.

Nanoparticles for Protein Sensing in Primary Containers: Interaction Analysis and Application.

PubMed

Pérez Medina Martínez, Víctor; Espinosa-de la Garza, Carlos E; Méndez-Silva, Diego A; Bolívar-Vichido, Mariana; Flores-Ortiz, Luis F; Pérez, Néstor O

2018-05-01

Silver nanoparticles (AgNPs) are known to interact with proteins, leading to modifications of the plasmonic absorption that can be used to monitor this interaction, entailing a promising application for sensing adsorption of therapeutic proteins in primary containers. First, transmission electron microscopy in combination with plasmonic absorption and light scattering responses were used to characterize AgNPs and protein-AgNP complexes, including its concentration dependence, using two therapeutic molecules as models: a monoclonal antibody (mAb) and a synthetic copolymer (SC). Upon interaction, a protein corona was formed around AgNPs with the consequent shifting and broadening of their characteristic surface plasmon resonance (SPR) band (400 nm) to 410 nm and longer wavelenghts. Additional studies revealed secondary and three-dimensional structure modifications of model proteins upon interaction with AgNPs by circular dichroism and fluorescence techniques, respectively. Based on the modification of the SPR condition of AgNPs upon interaction with proteins, we developed a novel protein-sensing application of AgNPs in primary containers. This strategy was used to conduct a compatibility assessment of model proteins towards five commercially available prefillable glass syringe (PFS) models. mAb- and SC-exposed PFSs showed that 74 and 94% of cases were positive for protein adsorption, respectively. Interestingly, protein adsorption on 15% of total tested PFSs was negligible (below the nanogram level). Our results highlight the need of a case-by-case compatibility assessment of therapeutic proteins and their primary containers. This strategy has the potential to be easily applied on other containers and implemented during early-stage product development by pharmaceutical companies and for routine use during batch release by packaging manufacturers.

Preferential Nucleosome Assembly at DNA Triplet Repeats from the Myotonic Dystrophy Gene

NASA Astrophysics Data System (ADS)

Wang, Yuh-Hwa; Amirhaeri, Sorour; Kang, Seongman; Wells, Robert D.; Griffith, Jack D.

1994-07-01

The expansion of CTG repeats in DNA occurs in or near genes involved in several human diseases, including myotonic dystrophy and Huntington's disease. Nucleosomes, the basic structural element of chromosomes, consist of 146 base pairs of DNA coiled about an octamer of histone proteins and mediate general transcriptional repression. Electron microscopy was used to examine in vitro the nucleosome assembly of DNA containing repeating CTG triplets. The efficiency of nucleosome formation increased with expanded triplet blocks, suggesting that such blocks may repress transcription through the creation of stable nucleosomes.

High-Resolution Mapping of a Repeat Protein Folding Free Energy Landscape.

PubMed

Fossat, Martin J; Dao, Thuy P; Jenkins, Kelly; Dellarole, Mariano; Yang, Yinshan; McCallum, Scott A; Garcia, Angel E; Barrick, Doug; Roumestand, Christian; Royer, Catherine A

2016-12-06

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the conformational ensemble along the entire folding reaction coordinate. Simulations can provide this level of insight for small proteins. In contrast, with the exception of hydrogen exchange, which does not monitor folding directly, experimental studies of protein folding have not yielded such structural and energetic detail. NMR can provide residue specific atomic level structural information, but its implementation in protein folding studies using chemical or temperature perturbation is problematic. Here we present a highly detailed structural and energetic map of the entire folding landscape of the leucine-rich repeat protein, pp32 (Anp32), obtained by combining pressure-dependent site-specific 1 H- 15 N HSQC data with coarse-grained molecular dynamics simulations. The results obtained using this equilibrium approach demonstrate that the main barrier to folding of pp32 is quite broad and lies near the unfolded state, with structure apparent only in the C-terminal region. Significant deviation from two-state unfolding under pressure reveals an intermediate on the folded side of the main barrier in which the N-terminal region is disordered. A nonlinear temperature dependence of the population of this intermediate suggests a large heat capacity change associated with its formation. The combination of pressure, which favors the population of folding intermediates relative to chemical denaturants; NMR, which allows their observation; and constrained structure-based simulations yield unparalleled insight into protein folding mechanisms. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation.

PubMed

Casillas-Ituarte, Nadia N; Cruz, Carlos H B; Lins, Roberto D; DiBartola, Alex C; Howard, Jessica; Liang, Xiaowen; Höök, Magnus; Viana, Isabelle F T; Sierra-Hernández, M Roxana; Lower, Steven K

2017-05-26

The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands ( e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/ k off ) and dissociation constants ( K d = k off / k on ), determined by mechanically dissociating the Fn·peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 μm) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 μm). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I ( K d app = 0.2-0.5 μm, as determined by SPR) compared with the lowest-affinity double-alanine peptide ( K d app = 3.8 μm). Together, these findings demonstrate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins.

PubMed

Gainer, Harold; House, Shirley; Kim, Dong Sun; Chin, Hemin; Pant, Harish C

2017-04-01

When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper, we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein are present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon's neuronal cell bodies, but despite the presence of NF mRNA in the giant axon no labeled NF protein is detected in the giant axon. This lends support to the glia-axon protein transfer hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.

Influence of casein as a percentage of true protein and protein level on color and texture of milks containing 1 and 2% fat.

PubMed

Misawa, Noriko; Barbano, David M; Drake, MaryAnne

2016-07-01

Combinations of fresh liquid microfiltration retentate of skim milk, ultrafiltered retentate and permeate produced from microfiltration permeate, cream, and dried lactose monohydrate were used to produce a matrix of 20 milks. The milks contained 5 levels of casein as a percentage of true protein of about 5, 25, 50, 75, and 80% and 4 levels of true protein of 3.0, 3.76, 4.34, and 5.0% with constant lactose percentage of 5%. The experiment was replicated twice and repeated for both 1 and 2% fat content. Hunter color measurements, relative viscosity, and fat globule size distribution were measured, and a trained panel documented appearance and texture attributes on all milks. Overall, casein as a percentage of true protein had stronger effects than level of true protein on Hunter L, a, b values, relative viscosity, and fat globule size when using fresh liquid micellar casein concentrates and milk serum protein concentrates produced by a combination of microfiltration and ultrafiltration. As casein as a percentage of true protein increased, the milks became more white (higher L value), less green (lower negative a value), and less yellow (lower b value). Relative viscosity increased and d(0.9) generally decreased with increasing casein as a percentage of true protein. Panelists perceived milks with increasing casein as a percentage of true protein as more white, more opaque, and less yellow. Panelists were able to detect increased throat cling and mouthcoating with increased casein as a percentage of true protein in 2% milks, even when differences in appearance among milks were masked. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Differential interaction and aggregation of 3-repeat and 4-repeat tau isoforms with 14-3-3{zeta} protein

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

Sadik, Golam; Tanaka, Toshihisa, E-mail: tanaka@psy.med.osaka-u.ac.jp; Kato, Kiyoko

2009-05-22

Tau isoforms, 3-repeat (3R) and 4-repeat tau (4R), are differentially involved in neuronal development and in several tauopathies. 14-3-3 protein binds to tau and 14-3-3/tau association has been found both in the development and in tauopathies. To understand the role of 14-3-3 in the differential regulation of tau isoforms, we have performed studies on the interaction and aggregation of 3R-tau and 4R-tau, either phosphorylated or unphosphorylated, with 14-3-3{zeta}. We show by surface plasmon resonance studies that the interaction between unphosphorylated 3R-tau and 14-3-3{zeta} is {approx}3-folds higher than that between unphosphorylated 4R-tau and 14-3-3{zeta}. Phosphorylation of tau by protein kinase Amore » (PKA) increases the affinity of both 3R- and 4R-tau for 14-3-3{zeta} to a similar level. An in vitro aggregation assay employing both transmission electron microscopy and fluorescence spectroscopy revealed the aggregation of unphosphorylated 4R-tau to be significantly higher than that of unphosphorylated 3R-tau following the induction of 14-3-3{zeta}. The filaments formed from 3R- and 4R-tau were almost similar in morphology. In contrast, the aggregation of both 3R- and 4R-tau was reduced to a similar low level after phosphorylation with PKA. Taken together, these results suggest that 14-3-3{zeta} exhibits a similar role for tau isoforms after PKA-phosphorylation, but a differential role for unphosphorylated tau. The significant aggregation of 4R-tau by 14-3-3{zeta} suggests that 14-3-3 may act as an inducer in the generation of 4R-tau-predominant neurofibrillary tangles in tauopathies.« less

Group B streptococcal serine-rich repeat proteins promote interaction with fibrinogen and vaginal colonization.

PubMed

Wang, Nai-Yu; Patras, Kathryn A; Seo, Ho Seong; Cavaco, Courtney K; Rösler, Berenice; Neely, Melody N; Sullam, Paul M; Doran, Kelly S

2014-09-15

Group B streptococcus (GBS) can cause severe disease in susceptible hosts, including newborns, pregnant women, and the elderly. GBS serine-rich repeat (Srr) surface glycoproteins are important adhesins/invasins in multiple host tissues, including the vagina. However, exact molecular mechanisms contributing to their importance in colonization are unknown. We have recently determined that Srr proteins contain a fibrinogen-binding region (BR) and hypothesize that Srr-mediated fibrinogen binding may contribute to GBS cervicovaginal colonization. In this study, we observed that fibrinogen enhanced wild-type GBS attachment to cervical and vaginal epithelium, and that this was dependent on Srr1. Moreover, purified Srr1-BR peptide bound directly to host cells, and peptide administration in vivo reduced GBS recovery from the vaginal tract. Furthermore, a GBS mutant strain lacking only the Srr1 "latching" domain exhibited decreased adherence in vitro and decreased persistence in a mouse model of GBS vaginal colonization, suggesting the importance of Srr-fibrinogen interactions in the female reproductive tract. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

[Polymorphic loci and polymorphism analysis of short tandem repeats within XNP gene].

PubMed

Liu, Qi-Ji; Gong, Yao-Qin; Guo, Chen-Hong; Chen, Bing-Xi; Li, Jiang-Xia; Guo, Yi-Shou

2002-01-01

To select polymorphic short tandem repeat markers within X-linked nuclear protein (XNP) gene, genomic clones which contain XNP gene were recognized by homologous analysis with XNP cDNA. By comparing the cDNA with genomic DNA, non-exonic sequences were identified, and short tandem repeats were selected from non-exonic sequences by using BCM search Launcher. Polymorphisms of the short tandem repeats in Chinese population were evaluated by PCR amplification and PAGE. Five short tandem repeats were identified from XNP gene, two of which were polymorphic. Four and 11 alleles were observed in Chinese population for XNPSTR1 and XNPSTR4, respectively. Heterozygosities were 47% for XNPSTR1 and 70% for XNPSTR4. XNPSTR1 and XNPSTR4 localized within 3' end and intron 10, respectively. Two polymorphic short tandem repeats have been identified within XNP gene and will be useful for linkage analysis and gene diagnosis of XNP gene.

Repeatability of two-dimensional chemical shift imaging multivoxel proton magnetic resonance spectroscopy for measuring human cerebral choline-containing compounds.

PubMed

Puri, Basant K; Egan, Mary; Wallis, Fintan; Jakeman, Philip

2018-03-22

To investigate the repeatability of proton magnetic resonance spectroscopy in the in vivo measurement of human cerebral levels of choline-containing compounds (Cho). Two consecutive scans were carried out in six healthy resting subjects at a magnetic field strength of 1.5 T. On each occasion, neurospectroscopy data were collected from 64 voxels using the same 2D chemical shift imaging (CSI) sequence. The data were analyzed in the same way, using the same software, to obtain the values for each voxel of the ratio of Cho to creatine. The Wilcoxon related-samples signed-rank test, coefficient of variation (CV), repeatability coefficient (RC), and intraclass correlation coefficient (ICC) were used to assess the repeatability. The CV ranged from 2.75% to 33.99%, while the minimum RC was 5.68%. There was excellent reproducibility, as judged by significant ICC values, in 26 voxels. Just three voxels showed significant differences according to the Wilcoxon related-samples signed-rank test. It is therefore concluded that when CSI multivoxel proton neurospectroscopy is used to measure cerebral choline-containing compounds at 1.5 T, the reproducibility is highly acceptable.

Purified horse milk exosomes contain an unpredictable small number of major proteins.

PubMed

Sedykh, Sergey E; Purvinish, Lada V; Monogarov, Artem S; Burkova, Evgeniya E; Grigor'eva, Alina E; Bulgakov, Dmitrii V; Dmitrenok, Pavel S; Vlassov, Valentin V; Ryabchikova, Elena I; Nevinsky, Georgy A

2017-06-01

Exosomes are 40-100 nm nanovesicles containing RNA and different proteins. Exosomes containing proteins, lipids, mRNAs, and microRNAs are important in intracellular communication and immune function. Exosomes from different sources are usually obtained by combination of centrifugation and ultracentrifugation and according to published data can contain from a few dozens to thousands of different proteins. Crude exosome preparations from milk of eighteen horses were obtained for the first time using several standard centrifugations. Exosome preparations were additionally purified by FPLC gel filtration. Individual preparations demonstrated different profiles of gel filtration showing well or bad separation of exosome peaks and one or two peaks of co-isolating proteins and their complexes. According to the electron microscopy, well purified exosomes displayed a typical exosome-like size (30-100 nm) and morphology. It was shown that exosomes may have several different biological functions, but detection of their biological functions may vary significantly depending on the presence of exosome contaminating proteins and proteins directly into exosomes. Exosome proteins were identified before and after gel filtration by MALDI MS and MS/MS spectrometry of protein tryptic hydrolyzates derived by SDS PAGE and 2D electrophoresis. The results of protein identification were unexpected: one or two peaks co-isolating proteins after gel-filtration mainly contained kappa-, beta-, alpha-S1-caseins and its precursors, but these proteins were not found in well-purified exosomes. Well-purified exosomes contained from five to eight different major proteins: CD81, CD63 receptors, beta-lactoglobulin and lactadherin were common to all preparations, while actin, butyrophilin, lactoferrin, and xanthine dehydrogenase were found only in some of them. The article describes the morphology and the protein content of major horse milk exosomes for the first time. Our results on the decrease of

Structure and Function of Na+-Symporters with Inverted Repeats

PubMed Central

Abramson, Jeff; Wright, Ernest M.

2009-01-01

Summary Symporters are membrane proteins that couple energy stored in electrochemical potential gradients to drive the cotransport of molecules and ions into cells. Traditionally, proteins are classified into gene families based on sequence homology and functional properties, e.g. the sodium glucose (SLC5 or Sodium Solute Symporter Family, SSS or SSF) and GABA (SLC6 or Neurotransmitter Sodium Symporter Family, NSS or SNF) symporter families [1-4]. Recently, it has been established that four Na+-symporter proteins with unrelated sequences have a common structural core containing an inverted repeat of 5 transmembrane (TM) helices [5-8]. Analysis of these four structures reveals that they reside in different conformations along the transport cycle providing atomic insight into the mechanism of sodium solute cotransport. PMID:19631523

Invasive Species Management on Military Lands: Clustered Regularly Interspaced Short Palindromic Repeat/ CRISPR associated protein 9 (CRISPR/Cas9) based Gene Drives

DTIC Science & Technology

2017-06-30

Clustered Regularly Interspaced Short Palindromic Repeat/ CRISPR -associated protein 9 ( CRISPR /Cas9)-based Gene Drives En vi ro nm en ta l L ab or at...Management on Military Lands Clustered Regularly Interspaced Short Palindromic Repeat/ CRISPR -associated protein 9 ( CRISPR /Cas9)-based Gene Drives Ping... CRISPR /Cas9-based Gene Drives for Invasive Species Management on Military Lands” ERDC/EL SR-17-2 ii Abstract Applications of genetic engineering

RRW: repeated random walks on genome-scale protein networks for local cluster discovery

PubMed Central

Macropol, Kathy; Can, Tolga; Singh, Ambuj K

2009-01-01

Background We propose an efficient and biologically sensitive algorithm based on repeated random walks (RRW) for discovering functional modules, e.g., complexes and pathways, within large-scale protein networks. Compared to existing cluster identification techniques, RRW implicitly makes use of network topology, edge weights, and long range interactions between proteins. Results We apply the proposed technique on a functional network of yeast genes and accurately identify statistically significant clusters of proteins. We validate the biological significance of the results using known complexes in the MIPS complex catalogue database and well-characterized biological processes. We find that 90% of the created clusters have the majority of their catalogued proteins belonging to the same MIPS complex, and about 80% have the majority of their proteins involved in the same biological process. We compare our method to various other clustering techniques, such as the Markov Clustering Algorithm (MCL), and find a significant improvement in the RRW clusters' precision and accuracy values. Conclusion RRW, which is a technique that exploits the topology of the network, is more precise and robust in finding local clusters. In addition, it has the added flexibility of being able to find multi-functional proteins by allowing overlapping clusters. PMID:19740439

Short intronic repeat sequences facilitate circular RNA production.

PubMed

Liang, Dongming; Wilusz, Jeremy E

2014-10-15

Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery "backsplices" and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼ 30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3' end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA. © 2014 Liang and Wilusz; Published by Cold Spring Harbor Laboratory Press.

Short intronic repeat sequences facilitate circular RNA production

PubMed Central

Liang, Dongming

2014-01-01

Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery “backsplices” and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3′ end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA. PMID:25281217

An update on polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein that protects crop plants against pathogens

PubMed Central

Kalunke, Raviraj M.; Tundo, Silvio; Benedetti, Manuel; Cervone, Felice; De Lorenzo, Giulia; D'Ovidio, Renato

2015-01-01

Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the pectin-depolymerizing activity of polygalacturonases secreted by microbial pathogens and insects. These ubiquitous inhibitors have a leucine-rich repeat structure that is strongly conserved in monocot and dicot plants. Previous reviews have summarized the importance of PGIP in plant defense and the structural basis of PG-PGIP interaction; here we update the current knowledge about PGIPs with the recent findings on the composition and evolution of pgip gene families, with a special emphasis on legume and cereal crops. We also update the information about the inhibition properties of single pgip gene products against microbial PGs and the results, including field tests, showing the capacity of PGIP to protect crop plants against fungal, oomycetes and bacterial pathogens. PMID:25852708

Aggregation landscapes of Huntingtin exon 1 protein fragments and the critical repeat length for the onset of Huntington’s disease

PubMed Central

Chen, Mingchen; Wolynes, Peter G.

2017-01-01

Huntington’s disease (HD) is a neurodegenerative disease caused by an abnormal expansion in the polyglutamine (polyQ) track of the Huntingtin (HTT) protein. The severity of the disease depends on the polyQ repeat length, arising only in patients with proteins having 36 repeats or more. Previous studies have shown that the aggregation of N-terminal fragments (encoded by HTT exon 1) underlies the disease pathology in mouse models and that the HTT exon 1 gene product can self-assemble into amyloid structures. Here, we provide detailed structural mechanisms for aggregation of several protein fragments encoded by HTT exon 1 by using the associative memory, water-mediated, structure and energy model (AWSEM) to construct their free energy landscapes. We find that the addition of the N-terminal 17-residue sequence (NT17) facilitates polyQ aggregation by encouraging the formation of prefibrillar oligomers, whereas adding the C-terminal polyproline sequence (P10) inhibits aggregation. The combination of both terminal additions in HTT exon 1 fragment leads to a complex aggregation mechanism with a basic core that resembles that found for the aggregation of pure polyQ repeats using AWSEM. At the extrapolated physiological concentration, although the grand canonical free energy profiles are uphill for HTT exon 1 fragments having 20 or 30 glutamines, the aggregation landscape for fragments with 40 repeats has become downhill. This computational prediction agrees with the critical length found for the onset of HD and suggests potential therapies based on blocking early binding events involving the terminal additions to the polyQ repeats. PMID:28400517

Immunogenicity of therapeutic proteins. Part 2: impact of container closures.

PubMed

Sharma, Basant

2007-01-01

Immunogenicity as a potential consequence of therapeutic protein administration is increasingly being scrutinized in the biopharmaceuticals industry, particularly with the imminent introduction of biosimilar products. Immunogenicity is an important safety aspect requiring rigorous investigation to fully appreciate its impact. Factors involved in product handling, such as storage temperature, light exposure, and shaking, have been implicated in immunogenicity, while container closure systems are no less important. Intended to provide a stable environment for the dosage form, container closures may also interact with a product, affecting performance and potentially enhancing immunogenicity. Glass surfaces, air-liquid interfaces, and lubricants can mediate protein denaturation, while phthalates in plastics and latex rubber are sources of extractables and leachates that may contaminate a product, causing allergic reactions and increasing immunogenicity. The manufacture of therapeutic proteins therefore requires rigorous safety evaluations not just in the context of the product, but also product containment.

Comprehensive analysis of all triple helical repeats in beta-spectrins reveals patterns of selective evolutionary conservation.

PubMed

Baines, Anthony J

2003-01-01

The spectrin superfamily (spectrin, alpha-actinin, utrophin and dystrophin) has in common a triple helical repeating unit of ~106 amino acid residues. In spectrin, alpha and beta chains contain multiple copies of this repeat. beta-spectrin chains contain the majority of binding activities in spectrin and are essential for animal life. Canonical beta-spectrins have 17 repeats; beta-heavy spectrins have 30. Here, the repeats of five human beta-spectrins, plus beta-spectrins from several other vertebrates and invertebrates, have been analysed. Repeats 1, 2, 14 and 17 in canonical beta are highly conserved between invertebrates and vertebrates, and repeat 8 in some isoforms. This is consistent with conservation of critical functions, since repeats 1, 2 and 17 bind alpha-spectrin. Repeats 1 of beta-spectrins are not always detected by SMART or Pfam tools. A profile hidden Markov model of beta-spectrin repeat 1 detects alpha-actinins, but not utrophin or dystrophin. Novel examples of repeat 1 were detected in the spectraplakins MACF1, BPAG1 and plectin close to the actin-binding domain. Ankyrin binds to the C-terminal portion of repeat 14; the high conservation of this entire repeat may point to additional, undiscovered ligand-binding activities. This analysis indicates that the basic triple helical repeat pattern was adapted early in the evolution of the spectrin superfamily to encompass essential binding activities, which characterise individual repeats in proteins extant today.

The Candidate Phylum Poribacteria by Single-Cell Genomics: New Insights into Phylogeny, Cell-Compartmentation, Eukaryote-Like Repeat Proteins, and Other Genomic Features

PubMed Central

Kamke, Janine; Rinke, Christian; Schwientek, Patrick; Mavromatis, Kostas; Ivanova, Natalia; Sczyrba, Alexander; Woyke, Tanja; Hentschel, Ute

2014-01-01

The candidate phylum Poribacteria is one of the most dominant and widespread members of the microbial communities residing within marine sponges. Cell compartmentalization had been postulated along with their discovery about a decade ago and their phylogenetic association to the Planctomycetes, Verrucomicrobia, Chlamydiae superphylum was proposed soon thereafter. In the present study we revised these features based on genomic data obtained from six poribacterial single cells. We propose that Poribacteria form a distinct monophyletic phylum contiguous to the PVC superphylum together with other candidate phyla. Our genomic analyses supported the possibility of cell compartmentalization in form of bacterial microcompartments. Further analyses of eukaryote-like protein domains stressed the importance of such proteins with features including tetratricopeptide repeats, leucin rich repeats as well as low density lipoproteins receptor repeats, the latter of which are reported here for the first time from a sponge symbiont. Finally, examining the most abundant protein domain family on poribacterial genomes revealed diverse phyH family proteins, some of which may be related to dissolved organic posphorus uptake. PMID:24498082

Simultaneous determination of phenolic acids and diterpenoids and their comparative pharmacokinetic study in normal and acute blood stasis rats by UFLC-MS/MS after oral administration of Guan-Xin-Shu-Tong capsules.

PubMed

Gao, Xun; Mu, Jingqing; Guan, Shaoyi; Li, Qing; Du, Yiyang; Zhang, Huifen; Bi, Kaishun

2018-01-01

Guan-Xin-Shu-Tong capsules are one of the well-known and first-line Chinese traditional herbal formula for treating coronary heart disease. A validated and sensitive method via ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) was established to simultaneously determinate five phenolic acids and four diterpenoids in rats in order to investigate their pharmacokinetic profiles firstly. Analytes were extracted by ethyl acetate and determined via multiple reaction monitoring mode in both positive and negative ion modes. The values for limit of quantification were in range of 0.025-1.250ng/ml. Inter- and intra-day precisions were no more than 10.9% with accuracy of -11.0%-10.6%, meanwhile the stable and suitable extraction recoveries were also obtained. And finally such excellent method was used to compare the pharmacokinetics of nine compounds in normal and acute blood stasis rats after oral administration of Guan-Xin-Shu-Tong capsules. Copyright © 2017 Elsevier B.V. All rights reserved.

Behavior of hollow balls containing granules bouncing repeatedly off the ground

NASA Astrophysics Data System (ADS)

Hu, Min; Mu, Qing-song; Luo, Ning; Li, Gang; Peng, Ning-bo

2013-07-01

An experimental study of the behavior of hollow balls filled with some granules (mung beans or millets) bouncing repeatedly off a static flat horizontal surface is presented. We observed that the bounce number of the ball is limited and decreases regularly with an increasing number of granules. Moreover, for two balls containing a different kind of granules, their bounce numbers are basically equal when the two balls have the same mass of granules. While there is no clear relationship between the first rebound height of one ball and the number of granules, there appears an exponential decay of the second rebound height with an increase of the granule number. Furthermore, a two-dimensional numerical model has been created to find out the law of the ball's rebound height and the dissipation law of the granule nested system. A generalized prediction equation to reasonably explain the law of the bounce number has also been proposed.

Immunogenicity of Recombinant Proteins Consisting of Plasmodium vivax Circumsporozoite Protein Allelic Variant-Derived Epitopes Fused with Salmonella enterica Serovar Typhimurium Flagellin

PubMed Central

Leal, Monica Teixeira Andrade; Camacho, Ariane Guglielmi Ariza; Teixeira, Laís Helena; Bargieri, Daniel Youssef; Soares, Irene Silva; Tararam, Cibele Aparecida

2013-01-01

A Plasmodium falciparum circumsporozoite protein (CSP)-based recombinant fusion vaccine is the first malaria vaccine to reach phase III clinical trials. Resistance to infection correlated with the production of antibodies to the immunodominant central repeat region of the CSP. In contrast to P. falciparum, vaccine development against the CSP of Plasmodium vivax malaria is far behind. Based on this gap in our knowledge, we generated a recombinant chimeric protein containing the immunodominant central repeat regions of the P. vivax CSP fused to Salmonella enterica serovar Typhimurium-derived flagellin (FliC) to activate the innate immune system. The recombinant proteins that were generated contained repeat regions derived from each of the 3 different allelic variants of the P. vivax CSP or a fusion of regions derived from each of the 3 allelic forms. Mice were subcutaneously immunized with the fusion proteins alone or in combination with the Toll-like receptor 3 (TLR-3) agonist poly(I·C), and the anti-CSP serum IgG response was measured. Immunization with a mixture of the 3 recombinant proteins, each containing immunodominant epitopes derived from a single allelic variant, rather than a single recombinant protein carrying a fusion of regions derived from each of 3 allelic forms elicited a stronger immune response. This response was independent of TLR-4 but required TLR-5/MyD88 activation. Antibody titers significantly increased when poly(I·C) was used as an adjuvant with a mixture of the 3 recombinant proteins. These recombinant fusion proteins are novel candidates for the development of an effective malaria vaccine against P. vivax. PMID:23863502

Revisiting the TALE repeat.

PubMed

Deng, Dong; Yan, Chuangye; Wu, Jianping; Pan, Xiaojing; Yan, Nieng

2014-04-01

Transcription activator-like (TAL) effectors specifically bind to double stranded (ds) DNA through a central domain of tandem repeats. Each TAL effector (TALE) repeat comprises 33-35 amino acids and recognizes one specific DNA base through a highly variable residue at a fixed position in the repeat. Structural studies have revealed the molecular basis of DNA recognition by TALE repeats. Examination of the overall structure reveals that the basic building block of TALE protein, namely a helical hairpin, is one-helix shifted from the previously defined TALE motif. Here we wish to suggest a structure-based re-demarcation of the TALE repeat which starts with the residues that bind to the DNA backbone phosphate and concludes with the base-recognition hyper-variable residue. This new numbering system is consistent with the α-solenoid superfamily to which TALE belongs, and reflects the structural integrity of TAL effectors. In addition, it confers integral number of TALE repeats that matches the number of bound DNA bases. We then present fifteen crystal structures of engineered dHax3 variants in complex with target DNA molecules, which elucidate the structural basis for the recognition of bases adenine (A) and guanine (G) by reported or uncharacterized TALE codes. Finally, we analyzed the sequence-structure correlation of the amino acid residues within a TALE repeat. The structural analyses reported here may advance the mechanistic understanding of TALE proteins and facilitate the design of TALEN with improved affinity and specificity.

New partner proteins containing novel internal recognition motif for human Glutaminase Interacting Protein (hGIP)

PubMed Central

Zencir, Sevil; Banerjee, Monimoy; Dobson, Melanie J.; Ayaydin, Ferhan; Fodor, Elfrieda Ayaydin; Topcu, Zeki; Mohanty, Smita

2013-01-01

Regulation of gene expression in cells is mediated by protein-protein, DNA-protein and receptor-ligand interactions. PDZ (PSD-95/Discs-large/ZO-1) domains are protein–protein interaction modules. PDZ-containing proteins function in the organization of multi-protein complexes controlling spatial and temporal fidelity of intracellular signaling pathways. In general, PDZ proteins possess multiple domains facilitating distinct interactions. The human Glutaminase Interacting Protein (hGIP) is an unusual PDZ protein comprising entirely of a single PDZ domain and plays pivotal roles in many cellular processes through its interaction with the C-terminus of partner proteins. Here, we report the identification by yeast two-hybrid screening of two new hGIP-interacting partners, DTX1 and STAU1. Both proteins lack the typical C-terminal PDZ recognition motif but contain a novel internal hGIP recognition motif recently identified in a phage display library screen. Fluorescence resonance energy transfer and confocal microscopy analysis confirmed the in vivo association of hGIP with DTX1 and STAU1 in mammalian cells validating the previous discovery of S/T-X-V/L-D as a consensus internal motif for hGIP recognition. Similar to hGIP, DTX1 and STAU1 have been implicated in neuronal function. Identification of these new interacting partners furthers our understanding of GIP-regulated signaling cascades and these interactions may represent potential new drug targets in humans. PMID:23395680

Repeat region of Brugia malayi sheath protein (Shp-1) carries Dominant B epitopes recognized in filarial endemic population.

PubMed

Jawaharlal, Jeya Prita Parasurama; Madhumathi, Jayaprakasam; Prince, Rajaiah Prabhu; Kaliraj, Perumal

2014-09-01

Transmission of lymphatic filariasis is mediated through microfilariae (L1 stage of the parasite) which is encased in an eggshell called sheath. The sheath protein Shp-1 stabilizes the structure due to the unique repeat region with Met-Pro-Pro-Gln-Gly sequences. Microfilarial proteins could be used as transmission blocking vaccines. Since the repeat region of Shp-1 was predicted to carry putative B epitopes, this region was used to analyze its reactivity with clinical samples towards construction of peptide vaccine. In silico analysis of Shp-1 showed the presence of B epitopes in the region 49-107. The polypeptide epitopic region Shp-149-107 was cloned and expressed in Escherichia coli. Antibody reactivity of the Shp-149-107 construct was evaluated in filarial endemic population by ELISA. Putatively immune endemic normals (EN) showed significantly high reactivity (P < 0.05) when compared to all the other categories. Antibody reactivity of Shp-1 repeat region was similar to that of whole protein proving that this region carries B epitopes responsible for its humoral response in humans. Thus this can be employed for inducing anti-microfilarial immunity in the infected population that may lead to reduction in transmission intensity and also it could be used along with other epitopes from different stages of the parasite in order to manage the disease effectively.

Possible reduced penetrance of expansion of 44 to 47 CAG/CAA repeats in the TATA-binding protein gene in spinocerebellar ataxia type 17.

PubMed

Oda, Masaya; Maruyama, Hirofumi; Komure, Osamu; Morino, Hiroyuki; Terasawa, Hideo; Izumi, Yuishin; Imamura, Tohru; Yasuda, Minoru; Ichikawa, Keiji; Ogawa, Masafumi; Matsumoto, Masayasu; Kawakami, Hideshi

2004-02-01

Spinocerebellar ataxia type 17 (SCA17) is an autosomal dominant cerebellar ataxia caused by expansion of CAG/CAA trinucleotide repeats in the TATA-binding protein (TBP) gene. Because the number of triplets in patients with SCA17 in previous studies ranged from 43 to 63, the normal number of trinucleotide units has been considered to be 42 or less. However, some healthy subjects in SCA17 pedigrees carry alleles with the same number of expanded repeats as patients with SCA17. To investigate the minimum number of CAG/CAA repeats in the TBP gene that causes SCA17. We amplified the region of the TBP gene containing the CAG/CAA repeat by means of polymerase chain reaction and performed fragment and sequence analyses. The subjects included 734 patients with SCA (480 patients with sporadic SCA and 254 patients with familial SCA) without CAG repeat expansions at the SCA1, SCA2, Machado-Joseph disease, SCA6, SCA7, or dentatorubral-pallidolluysian atrophy loci, with 162 healthy subjects, 216 patients with Parkinson disease, and 195 with Alzheimer disease as control subjects. Eight patients with SCA possessed an allele with more than 43 CAG/CAA repeats. Among the non-SCA groups, alleles with 43 to 45 repeats were seen in 3 healthy subjects and 2 with Parkinson disease. In 1 SCA pedigree, a patient with possible SCA17 and her healthy sister had alleles with 45 repeats. A 34-year-old man carrying alleles with 47 and 44 repeats (47/44) had developed progressive cerebellar ataxia and myoclonus at 25 years of age, and he exhibited dementia and pyramidal signs. He was the only affected person in his pedigree, although his father and mother carried alleles with mildly expanded repeats (44/36 and 47/36, respectively). In another pedigree, 1 patient carried a 43-repeat allele, whereas another patient had 2 normal alleles, indicating that the 43-repeat allele may not be pathologic in this family. We estimate that 44 CAG/CAA repeats is the minimum number required to cause SCA17. However

Predicting repeat protein folding kinetics from an experimentally determined folding energy landscape

PubMed Central

Street, Timothy O; Barrick, Doug

2009-01-01

The Notch ankyrin domain is a repeat protein whose folding has been characterized through equilibrium and kinetic measurements. In previous work, equilibrium folding free energies of truncated constructs were used to generate an experimentally determined folding energy landscape (Mello and Barrick, Proc Natl Acad Sci USA 2004;101:14102–14107). Here, this folding energy landscape is used to parameterize a kinetic model in which local transition probabilities between partly folded states are based on energy values from the landscape. The landscape-based model correctly predicts highly diverse experimentally determined folding kinetics of the Notch ankyrin domain and sequence variants. These predictions include monophasic folding and biphasic unfolding, curvature in the unfolding limb of the chevron plot, population of a transient unfolding intermediate, relative folding rates of 19 variants spanning three orders of magnitude, and a change in the folding pathway that results from C-terminal stabilization. These findings indicate that the folding pathway(s) of the Notch ankyrin domain are thermodynamically selected: the primary determinants of kinetic behavior can be simply deduced from the local stability of individual repeats. PMID:19177351

Molecular view of ligands specificity for CAG repeats in anti-Huntington therapy.

PubMed

Bochicchio, Anna; Rossetti, Giulia; Tabarrini, Oriana; Krauβ, Sybille; Carloni, Paolo

2015-10-13

Huntington's disease is a fatal and devastating neurodegenerative genetic disorder for which there is currently no cure. It is characterized by Huntingtin protein's mRNA transcripts with 36 or more CAG repeats. Inhibiting the formation of pathological complexes between these expanded transcripts and target proteins may be a valuable strategy against the disease. Yet, the rational design of molecules specifically targeting the expanded CAG repeats is limited by the lack of structural information. Here, we use well-tempered metadynamics-based free energy calculations to investigate pose and affinity of two ligands targeting CAG repeats for which affinities have been previously measured. The first consists of two 4-guanidinophenyl rings linked by an ester group. It is the most potent ligand identified so far, with Kd = 60(30) nM. The second consists of a 4-phenyl dihydroimidazole and 4-1H-indole dihydroimidazole connected by a C-C bond (Kd = 700(80) nM). Our calculations reproduce the experimental affinities and uncover the recognition pattern between ligands' and their RNA target. They also provide a molecular basis for the markedly different affinity of the two ligands for CAG repeats as observed experimentally. These findings may pave the way for a structure-based hit-to-lead optimization to further improve ligand selectivity toward CAG repeat-containing mRNAs.

Isolation and mapping of telomeric pentanucleotide (TAACC)n repeats of the Pacific whiteleg shrimp, Penaeus vannamei, using fluorescence in situ hybridization.

PubMed

Alcivar-Warren, Acacia; Meehan-Meola, Dawn; Wang, Yongping; Guo, Ximing; Zhou, Linghua; Xiang, Jianhai; Moss, Shaun; Arce, Steve; Warren, William; Xu, Zhenkang; Bell, Kireina

2006-01-01

To develop genetic and physical maps for shrimp, accurate information on the actual number of chromosomes and a large number of genetic markers is needed. Previous reports have shown two different chromosome numbers for the Pacific whiteleg shrimp, Penaeus vannamei, the most important penaeid shrimp species cultured in the Western hemisphere. Preliminary results obtained by direct sequencing of clones from a Sau3A-digested genomic library of P. vannamei ovary identified a large number of (TAACC/GGTTA)-containing SSRs. The objectives of this study were to (1) examine the frequency of (TAACC)n repeats in 662 P. vannamei genomic clones that were directly sequenced, and perform homology searches of these clones, (2) confirm the number of chromosomes in testis of P. vannamei, and (3) localize the TAACC repeats in P. vannamei chromosome spreads using fluorescence in situ hybridization (FISH). Results for objective 1 showed that 395 out of the 662 clones sequenced contained single or multiple SSRs with three or more repeat motifs, 199 of which contained variable tandem repeats of the pentanucleotide (TAACC/GGTTA)n, with 3 to 14 copies per sequence. The frequency of (TAACC)n repeats in P. vannamei is 4.68 kb for SSRs with five or more repeat motifs. Sequence comparisons using the BLASTN nonredundant and expressed sequence tag (EST) databases indicated that most of the TAACC-containing clones were similar to either the core pentanucleotide repeat in PVPENTREP locus (GenBank accession no. X82619) or portions of 28S rRNA. Transposable elements (transposase for Tn1000 and reverse transcriptase family members), hypothetical or unnamed protein products, and genes of known function such as 18S and 28S rRNAs, heat shock protein 70, and thrombospondin were identified in non-TAACC-containing clones. For objective 2, the meiotic chromosome number of P. vannamei was confirmed as N = 44. For objective 3, four FISH probes (P1 to P4) containing different numbers of TAACC repeats produced

Aberrant expression of epithelial leucine-rich repeat containing G protein-coupled receptor 5-positive cells in the eutopic endometrium in endometriosis and implications in deep-infiltrating endometriosis.

PubMed

Vallvé-Juanico, Júlia; Suárez-Salvador, Elena; Castellví, Josep; Ballesteros, Agustín; Taylor, Hugh S; Gil-Moreno, Antonio; Santamaria, Xavier

2017-11-01

To characterize leucine-rich repeat containing G protein-coupled receptor 5-positive (LGR5 + ) cells from the endometrium of women with endometriosis. Prospective experimental study. University hospital/fertility clinic. Twenty-seven women with endometriosis who underwent surgery and 12 healthy egg donors, together comprising 39 endometrial samples. Obtaining of uterine aspirates by using a Cornier Pipelle. Immunofluorescence in formalin-fixed paraffin-embedded tissue from mice and healthy and pathologic human endometrium using antibodies against LGR5, E-cadherin, and cytokeratin, and epithelial and stromal LGR5 + cells isolated from healthy and pathologic human eutopic endometrium by fluorescence-activated cell sorting and transcriptomic characterization by RNA high sequencing. Immunofluorescence showed that LGR5 + cells colocalized with epithelial markers in the stroma of the endometrium only in endometriotic patients. The results from RNA high sequencing of LGR5 + cells from epithelium and stroma did not show any statistically significant differences between them. The LGR5 + versus LGR5 - cells in pathologic endometrium showed 394 differentially expressed genes. The LGR5 + cells in deep-infiltrating endometriosis expressed inflammatory markers not present in the other types of the disease. Our results revealed the presence of aberrantly located LGR5 + cells coexpressing epithelial markers in the stromal compartment of women with endometriosis. These cells have a statistically significantly different expression profile in deep-infiltrating endometriosis in comparison with other types of endometriosis, independent of the menstrual cycle phase. Further studies are needed to elucidate their role and influence in reproductive outcomes. Copyright © 2017. Published by Elsevier Inc.

Wound induced Beta vulgaris polygalacturonase-inhibiting protein genes encode a longer leucine-rich repeat domain and inhibit fungal polygalacturonases

USDA-ARS?s Scientific Manuscript database

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defense. Sugar beet (Beta vulgaris L.) PGIP genes, BvPGIP1, BvPGIP2 and BvPGIP3, were isolated from two breeding lines, F1016 and F1010. Full-length cDNA sequences of the three BvPGIP genes encod...

Functions of bromodomain-containing proteins and their roles in homeostasis and cancer.

PubMed

Fujisawa, Takao; Filippakopoulos, Panagis

2017-04-01

Bromodomains (BRDs) are evolutionarily conserved protein-protein interaction modules that are found in a wide range of proteins with diverse catalytic and scaffolding functions and are present in most tissues. BRDs selectively recognize and bind to acetylated Lys residues - particularly in histones - and thereby have important roles in the regulation of gene expression. BRD-containing proteins are frequently dysregulated in cancer, they participate in gene fusions that generate diverse, frequently oncogenic proteins, and many cancer-causing mutations have been mapped to the BRDs themselves. Importantly, BRDs can be targeted by small-molecule inhibitors, which has stimulated many translational research projects that seek to attenuate the aberrant functions of BRD-containing proteins in disease.

Effect of competing self-structure on triplex formation with purine-rich oligodeoxynucleotides containing GA repeats.

PubMed Central

Noonberg, S B; François, J C; Garestier, T; Hélène, C

1995-01-01

Competition between triplex formation with double-stranded DNA and oligonucleotide self-association was investigated in 23mer GA and GT oligonucleotides containing d(GA)5 or d(GT)5 repeats. Whereas triplex formation with GT oligonucleotides was diminished when temperature increased from 4 to 37 degrees C, triplex formation with GA oligonucleotides was enhanced when temperature increased within the same range due to the presence of competing intermolecular GA oligonucleotide self-structure. This self-structure was determined to be a homoduplex stabilized by the internal GA repeats. UV spectroscopy of these homoduplexes demonstrated a single sharp transition with rapid kinetics (Tm = 38.5-43.5 degrees C over strand concentrations of 0.5-4 microM, respectively, with transition enthalpy, delta H = -89 +/- 7 kcal/mol) in 10 mM MgCl2, 100 mM NaCl, pH 7.0. Homoduplex formation was strongly stabilized by multivalent cations (spermine > Mg2+ = Ca2+) and destabilized by low concentrations of monovalent cations (K+ = Li+ = Na+) in the presence of divalent cations. However, unlike GA or GT oligonucleotide-containing triplexes, the homoduplex formed even in the absence of multivalent cations, stabilized by only moderate concentrations of monovalent cations (Li+ > Na+ > K+). Through the development of multiple equilibrium states and the resulting depletion of free oligonucleotide, it was found that the presence of competing self-structure could decrease triplex formation under a variety of experimental conditions. Images PMID:7596824

Instability of expanded CAG/CAA repeats in spinocerebellar ataxia type 17.

PubMed

Gao, Rui; Matsuura, Tohru; Coolbaugh, Mary; Zühlke, Christine; Nakamura, Koichiro; Rasmussen, Astrid; Siciliano, Michael J; Ashizawa, Tetsuo; Lin, Xi

2008-02-01

Trinucleotide repeat expansions are dynamic mutations causing many neurological disorders, and their instability is influenced by multiple factors. Repeat configuration seems particularly important, and pure repeats are thought to be more unstable than interrupted repeats. But direct evidence is still lacking. Here, we presented strong support for this hypothesis from our studies on spinocerebellar ataxia type 17 (SCA17). SCA17 is a typical polyglutamine disease caused by CAG repeat expansion in TBP (TATA binding protein), and is unique in that the pure expanded polyglutamine tract is coded by either a simple configuration with long stretches of pure CAGs or a complex configuration containing CAA interruptions. By small pool PCR (SP-PCR) analysis of blood DNA from SCA17 patients of distinct racial backgrounds, we quantitatively assessed the instability of these two types of expanded alleles coding similar length of polyglutamine expansion. Mutation frequency in patients harboring pure CAG repeats is 2-3 folds of those with CAA interruptions. Interestingly, the pure CAG repeats showed both expansion and deletion while the interrupted repeats exhibited mostly deletion at a significantly lower frequency. These data strongly suggest that repeat configuration is a critical determinant for instability, and CAA interruptions might serve as a limiting element for further expansion of CAG repeats in SCA17 locus, suggesting a molecular basis for lack of anticipation in SCA17 families with interrupted CAG expansion.

Identification and Expression Analysis of the Interferon-Induced Protein with Tetratricopeptide Repeats 5 (IFIT5) Gene in Duck (Anas platyrhynchos domesticus)

PubMed Central

Mu, Chunyu; Su, Yanhui; Liu, Ran; Huang, Zhengyang; Li, Yang; Yu, Qingming; Chang, Guobin; Xu, Qi; Chen, Guohong

2015-01-01

The interferon-induced proteins with tetratricopeptide repeats (IFITs) protein family mediates antiviral effects by inhibiting translation initiation, cell proliferation, and migration in the interferon (IFN) dependent innate immune system. Several members of this family, including IFIT1, IFIT2, IFIT3 and IFIT5, have been heavily studied in mammals. Avian species contain only one family member, IFIT5, and little is known about the role of this protein in birds. In this study, duck IFIT5 (duIFIT5) full-length mRNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE). Based on the sequence obtained, we performed a series of bioinformatics analyses, and found that duIFIT5 was most similar to homologs in other avian species. Also, duIFIT5 contained eight conserved TPR motifs and two conserved multi-domains (TPR_11 and TPR_12). Finally, we used duck hepatitis virus type 1 (DHV-1) and polyriboinosinicpolyribocytidylic acid (poly (I:C)) as a pathogen or a pathogen-associated molecular pattern induction to infect three-day-old domestic ducklings. The liver and spleen were collected to detect the change in duIFIT5 transcript level upon infection by quantitative real-time PCR (qRT-PCR). DuIFIT5 expression rapidly increased after DHV-1 infection and maintained a high level, while the transcripts of duIFIT5 peaked at 8h after poly (I:C) infection and then returned to normal. Taken together, these results provide a greater understanding of avian IFIT5. PMID:25816333

Serine 192 in the tiny RS repeat of the adenoviral L4-33K splicing enhancer protein is essential for function and reorganization of the protein to the periphery of viral replication centers

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

Oestberg, Sara, E-mail: sara.ostberg@imbim.uu.se; Toermaenen Persson, Heidi, E-mail: heidi.tormanen.persson@imbim.uu.se; Akusjaervi, Goeran, E-mail: goran.akusjarvi@imbim.uu.se

2012-11-25

The adenovirus L4-33K protein is a key regulator involved in the temporal shift from early to late pattern of mRNA expression from the adenovirus major late transcription unit. L4-33K is a virus-encoded alternative splicing factor, which enhances processing of 3 Prime splice sites with a weak sequence context. Here we show that L4-33K expressed from a plasmid is localized at the nuclear margin of uninfected cells. During an infection L4-33K is relocalized to the periphery of E2A-72K containing viral replication centers. We also show that serine 192 in the tiny RS repeat of the conserved carboxy-terminus of L4-33K, which ismore » critical for the splicing enhancer function of L4-33K, is necessary for the nuclear localization and redistribution of the protein to viral replication sites. Collectively, our results show a good correlation between the activity of L4-33K as a splicing enhancer protein and its localization to the periphery of viral replication centers.« less

Serine-Aspartate Repeat Protein D Increases Staphylococcus aureus Virulence and Survival in Blood.

PubMed

Askarian, Fatemeh; Uchiyama, Satoshi; Valderrama, J Andrés; Ajayi, Clement; Sollid, Johanna U E; van Sorge, Nina M; Nizet, Victor; van Strijp, Jos A G; Johannessen, Mona

2017-01-01

Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and infection. Deletion of sdrD from S. aureus subsp. aureus strain NCTC8325-4 attenuated bacterial survival in human whole blood ex vivo, which was associated with increased killing by human neutrophils. Remarkably, SdrD was able to inhibit innate immune-mediated bacterial killing independently of other S. aureus proteins, since addition of recombinant SdrD protein and heterologous expression of SdrD in Lactococcus lactis promoted bacterial survival in human blood. SdrD contributes to bacterial virulence in vivo, since fewer S. aureus subsp. aureus NCTC8325-4 ΔsdrD bacteria than bacteria of the parent strain were recovered from blood and several organs using a murine intravenous infection model. Collectively, our findings reveal a new property of SdrD as an important key contributor to S. aureus survival and the ability to escape the innate immune system in blood. Copyright © 2016 Askarian et al.

Serine-Aspartate Repeat Protein D Increases Staphylococcus aureus Virulence and Survival in Blood

PubMed Central

Uchiyama, Satoshi; Valderrama, J. Andrés; Ajayi, Clement; Sollid, Johanna U. E.; van Sorge, Nina M.; Nizet, Victor; van Strijp, Jos A. G.

2016-01-01

ABSTRACT Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and infection. Deletion of sdrD from S. aureus subsp. aureus strain NCTC8325-4 attenuated bacterial survival in human whole blood ex vivo, which was associated with increased killing by human neutrophils. Remarkably, SdrD was able to inhibit innate immune-mediated bacterial killing independently of other S. aureus proteins, since addition of recombinant SdrD protein and heterologous expression of SdrD in Lactococcus lactis promoted bacterial survival in human blood. SdrD contributes to bacterial virulence in vivo, since fewer S. aureus subsp. aureus NCTC8325-4 ΔsdrD bacteria than bacteria of the parent strain were recovered from blood and several organs using a murine intravenous infection model. Collectively, our findings reveal a new property of SdrD as an important key contributor to S. aureus survival and the ability to escape the innate immune system in blood. PMID:27795358

Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

PubMed

Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

1995-11-11

The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF.

PDZ-containing proteins: alternative splicing as a source of functional diversity.

PubMed

Sierralta, Jimena; Mendoza, Carolina

2004-12-01

Scaffold proteins allow specific protein complexes to be assembled in particular regions of the cell at which they organize subcellular structures and signal transduction complexes. This characteristic is especially important for neurons, which are highly polarized cells. Among the domains contained by scaffold proteins, the PSD-95, Discs-large, ZO-1 (PDZ) domains are of particular relevance in signal transduction processes and maintenance of neuronal and epithelial polarity. These domains are specialized in the binding of the carboxyl termini of proteins allowing membrane proteins to be localized by the anchoring to the cytoskeleton mediated by PDZ-containing scaffold proteins. In vivo studies carried out in Drosophila have taught that the role of many scaffold proteins is not limited to a single process; thus, in many cases the same genes are expressed in different tissues and participate in apparently very diverse processes. In addition to the differential expression of interactors of scaffold proteins, the expression of variants of these molecular scaffolds as the result of the alternative processing of the genes that encode them is proving to be a very important source of variability and complexity on a main theme. Alternative splicing in the nervous system is well documented, where specific isoforms play roles in neurotransmission, ion channel function, neuronal cell recognition, and are developmentally regulated making it a major mechanism of functional diversity. Here we review the current state of knowledge about the diversity and the known function of PDZ-containing proteins in Drosophila with emphasis in the role played by alternatively processed forms in the diversity of functions attributed to this family of proteins.

Fine tuning cellular recognition: The function of the leucine rich repeat (LRR) trans-membrane protein, LRT, in muscle targeting to tendon cells.

PubMed

Gilsohn, Eli; Volk, Talila

2010-01-01

The formation of complex tissues during embryonic development is often accompanied by directed cellular migration towards a target tissue. Specific mutual recognition between the migrating cell and its target tissue leads to the arrest of the cell migratory behavior and subsequent contact formation between the two interacting cell types. Recent studies implicated a novel family of surface proteins containing a trans-membrane domain and single leucine-rich repeat (LRR) domain in inter-cellular recognition and the arrest of cell migration. Here, we describe the involvement of a novel LRR surface protein, LRT, in targeting migrating muscles towards their corresponding tendon cells in the Drosophila embryo. LRT is specifically expressed by the target tendon cells and is essential for arresting the migratory behavior of the muscle cells. Additional studies in Drosophila S2 cultured cells suggest that LRT forms a protein complex with the Roundabout (Robo) receptor, essential for guiding muscles towards their tendon partners. Genetic analysis supports a model in which LRT performs its activity non-autonomously through its interaction with the Robo receptors expressed on the muscle surfaces. These results suggest a novel mechanism of intercellular recognition through interactions between LRR family members and Robo receptors.

Polyubiquitination of the B-cell translocation gene 1 and 2 proteins is promoted by the SCF ubiquitin ligase complex containing βTrCP.

PubMed

Sasajima, Hitoshi; Nakagawa, Koji; Kashiwayanagi, Makoto; Yokosawa, Hideyoshi

2012-01-01

B-cell translocation gene 1 and 2 (BTG1 and BTG2) are members of the BTG/Tob antiproliferative protein family, which is able to regulate the cell cycle and cell proliferation. We previously reported that BTG1, BTG2, Tob, and Tob2 are degraded via the ubiquitin-proteasome pathway. In this study, we investigated the mechanism of polyubiquitination of BTG1 and BTG2. Since the Skp1-Cdc53/Cullin 1-F-box protein (SCF) complex functions as one of the major ubiquitin ligases for cell cycle regulation, we first examined interactions between BTG proteins and components of the SCF complex, and found that BTG1 and BTG2 were capable of interacting with the SCF complex containing Cullin-1 (a scaffold protein) and Skp1 (a linker protein). As the SCF complex can ubiquitinate various target proteins by substituting different F-box proteins as subunits that recognize different target proteins, we next examined which F-box proteins could bind the two BTG proteins, and found that Skp2, β-transducin repeat-containing protein 1 (βTrCP1), and βTrCP2 were able to associate with both BTG1 and BTG2. Furthermore, we obtained evidence showing that βTrCP1 enhanced the polyubiquitination of both BTG1 and BTG2 more efficiently than Skp2 did, and that an F-box truncated mutant of βTrCP1 had a dominant negative effect on this polyubiquitination. Thus, we propose that BTG1 and BTG2 are subjected to polyubiquitination, more efficiently when it is mediated by SCFβTrCP than by SCFSkp2.

Repeat-associated plasticity in the Helicobacter pylori RD gene family.

PubMed

Shak, Joshua R; Dick, Jonathan J; Meinersmann, Richard J; Perez-Perez, Guillermo I; Blaser, Martin J

2009-11-01

The bacterium Helicobacter pylori is remarkable for its ability to persist in the human stomach for decades without provoking sterilizing immunity. Since repetitive DNA can facilitate adaptive genomic flexibility via increased recombination, insertion, and deletion, we searched the genomes of two H. pylori strains for nucleotide repeats. We discovered a family of genes with extensive repetitive DNA that we have termed the H. pylori RD gene family. Each gene of this family is composed of a conserved 3' region, a variable mid-region encoding 7 and 11 amino acid repeats, and a 5' region containing one of two possible alleles. Analysis of five complete genome sequences and PCR genotyping of 42 H. pylori strains revealed extensive variation between strains in the number, location, and arrangement of RD genes. Furthermore, examination of multiple strains isolated from a single subject's stomach revealed intrahost variation in repeat number and composition. Despite prior evidence that the protein products of this gene family are expressed at the bacterial cell surface, enzyme-linked immunosorbent assay and immunoblot studies revealed no consistent seroreactivity to a recombinant RD protein by H. pylori-positive hosts. The pattern of repeats uncovered in the RD gene family appears to reflect slipped-strand mispairing or domain duplication, allowing for redundancy and subsequent diversity in genotype and phenotype. This novel family of hypervariable genes with conserved, repetitive, and allelic domains may represent an important locus for understanding H. pylori persistence in its natural host.

Aggregation propensity of critical regions of the protein Tau

NASA Astrophysics Data System (ADS)

Muthee, Micaiah; Ahmed, Azka; Larini, Luca

The Alzheimer's disease is an irreversible, progressive brain disorder that slowly destroys memory and thinking skills, which eventually leads to the ability to not able to carry out the simplest tasks. The Alzheimer's disease is characterized by the formation of protein aggregates both within and outside of the brain's cells, the neurons. Within the neurons, the aggregation of the protein tau leads to the destruction of the microtubules in the axon of the neuron. Tau belongs to a group of proteins referred to as Microtubule-Associated Proteins. It is extremely flexible and is classified as an intrinsically unstructured protein due to its low propensity to form secondary structure. Tau promotes tubulin assembly into microtubules thereby stabilizing the cytoskeleton of the axon of the neurons. The microtubule binding region of tau consists of 4 pseudo-repeats. In this study, we will focus on the aggregation propensity of two fragments. In this study we will focus on the PHF43 fragment that contains the third pseudo-repeat and has been shown experimentally to aggregate readily. Another fragment that contains the second pseudo-repeat will be considered as well. Mutations in this region are associated with various form of dementia and for this reason we will consider the mutant P301L.

soc-2 encodes a leucine-rich repeat protein implicated in fibroblast growth factor receptor signaling

PubMed Central

Selfors, Laura M.; Schutzman, Jennifer L.; Borland, Christina Z.; Stern, Michael J.

1998-01-01

Activation of fibroblast growth factor (FGF) receptors elicits diverse cellular responses including growth, mitogenesis, migration, and differentiation. The intracellular signaling pathways that mediate these important processes are not well understood. In Caenorhabditis elegans, suppressors of clr-1 identify genes, termed soc genes, that potentially mediate or activate signaling through the EGL-15 FGF receptor. We demonstrate that three soc genes, soc-1, soc-2, and sem-5, suppress the activity of an activated form of the EGL-15 FGF receptor, consistent with the soc genes functioning downstream of EGL-15. We show that soc-2 encodes a protein composed almost entirely of leucine-rich repeats, a domain implicated in protein–protein interactions. We identified a putative human homolog, SHOC-2, which is 54% identical to SOC-2. We find that shoc-2 maps to 10q25, shoc-2 mRNA is expressed in all tissues assayed, and SHOC-2 protein is cytoplasmically localized. Within the leucine-rich repeats of both SOC-2 and SHOC-2 are two YXNX motifs that are potential tyrosine-phosphorylated docking sites for the SEM-5/GRB2 Src homology 2 domain. However, phosphorylation of these residues is not required for SOC-2 function in vivo, and SHOC-2 is not observed to be tyrosine phosphorylated in response to FGF stimulation. We conclude that this genetic system has allowed for the identification of a conserved gene implicated in mediating FGF receptor signaling in C. elegans. PMID:9618511

Interaction of Prevotella intermedia Strain 17 Leucine-Rich Repeat Domain Protein AdpF with Eukaryotic Cells Promotes Bacterial Internalization

PubMed Central

Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K.; Miyazaki, Hiroshi

2014-01-01

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells. PMID:24711565

Interaction of Prevotella intermedia strain 17 leucine-rich repeat domain protein AdpF with eukaryotic cells promotes bacterial internalization.

PubMed

Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K; Miyazaki, Hiroshi; Lewis, Janina P

2014-06-01

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.

The hydrophobic repeated domain of the Clostridium cellulovorans cellulose-binding protein (CbpA) has specific interactions with endoglucanases.

PubMed Central

Takagi, M; Hashida, S; Goldstein, M A; Doi, R H

1993-01-01

We overexpressed one of the hydrophobic repeated domains (HBDs) (110 amino acid residues) of the cellulose-binding protein (CbpA) from Clostridium cellulovorans by making a hybrid protein with the Escherichia coli maltose-binding protein (MalE). The HBD was purified to homogeneity, and interactions between the HBD and endoglucanases were analyzed by a novel interaction Western blotting (immunoblotting) method. The HBD had specific interactions with endoglucanases (EngB and EngD) from C. cellulovorans. These results indicated that the HBD was an endoglucanase binding site of CbpA. Images PMID:8226657

In silico analysis of Schmidtea mediterranea TIR domain-containing proteins.

PubMed

Tsoumtsa, Landry Laure; Sougoufora, Seynabou; Torre, Cedric; Lemichez, Emmanuel; Pontarotti, Pierre; Ghigo, Eric

2018-09-01

While genetic evidence points towards an absence of Toll-Like Receptors (TLRs) in Platyhelminthes, the Toll/IL-1 Receptor (TIR)-domains that drive the assembly of signalling complexes downstream TLR are present in these organisms. Here, we undertook the characterisation of the repertoire of TIR-domain containing proteins in Schmidtea mediterranea in order to gain valuable information on TLR evolution in metazoan. We report the presence of twenty proteins containing between one and two TIR domains. In addition, our phylogenetic-based reconstruction approach identified Smed-SARM and Smed-MyD88 as conserved TLR adaptors. Copyright © 2018 Elsevier Ltd. All rights reserved.

Large-scale modelling of the divergent spectrin repeats in nesprins: giant modular proteins.

PubMed

Autore, Flavia; Pfuhl, Mark; Quan, Xueping; Williams, Aisling; Roberts, Roland G; Shanahan, Catherine M; Fraternali, Franca

2013-01-01

Nesprin-1 and nesprin-2 are nuclear envelope (NE) proteins characterized by a common structure of an SR (spectrin repeat) rod domain and a C-terminal transmembrane KASH [Klarsicht-ANC-Syne-homology] domain and display N-terminal actin-binding CH (calponin homology) domains. Mutations in these proteins have been described in Emery-Dreifuss muscular dystrophy and attributed to disruptions of interactions at the NE with nesprins binding partners, lamin A/C and emerin. Evolutionary analysis of the rod domains of the nesprins has shown that they are almost entirely composed of unbroken SR-like structures. We present a bioinformatical approach to accurate definition of the boundaries of each SR by comparison with canonical SR structures, allowing for a large-scale homology modelling of the 74 nesprin-1 and 56 nesprin-2 SRs. The exposed and evolutionary conserved residues identify important pbs for protein-protein interactions that can guide tailored binding experiments. Most importantly, the bioinformatics analyses and the 3D models have been central to the design of selected constructs for protein expression. 1D NMR and CD spectra have been performed of the expressed SRs, showing a folded, stable, high content α-helical structure, typical of SRs. Molecular Dynamics simulations have been performed to study the structural and elastic properties of consecutive SRs, revealing insights in the mechanical properties adopted by these modules in the cell.

PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

PubMed

Dunn, Henry A; Ferguson, Stephen S G

2015-10-01

G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

Crystallization of a pentapeptide-repeat protein by reductive cyclic pentylation of free amines with glutaraldehyde

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

Vetting, Matthew W., E-mail: vetting@aecom.yu.edu; Hegde, Subray S.; Blanchard, John S.

2009-05-01

A method to modify proteins with glutaraldehyde under reducing conditions is presented. Treatment with glutaraldehyde and dimethylaminoborane was found to result in cyclic pentylation of free amines and facilitated the structural determination of a protein previously recalcitrant to the formation of diffraction quality crystals. The pentapeptide-repeat protein EfsQnr from Enterococcus faecalis protects DNA gyrase from inhibition by fluoroquinolones. EfsQnr was cloned and purified to homogeneity, but failed to produce diffraction-quality crystals in initial crystallization screens. Treatment of EfsQnr with glutaraldehyde and the strong reducing agent borane–dimethylamine resulted in a derivatized protein which produced crystals that diffracted to 1.6 Å resolution;more » their structure was subsequently determined by single-wavelength anomalous dispersion. Analysis of the derivatized protein using Fourier transform ion cyclotron resonance mass spectrometry indicated a mass increase of 68 Da per free amino group. Electron-density maps about a limited number of structurally ordered lysines indicated that the modification was a cyclic pentylation of free amines, producing piperidine groups.« less

Examination of the effect of the annealing cation on higher order structures containing guanine or isoguanine repeats

PubMed Central

Pierce, Sarah E.; Wang, Junmei; Jayawickramarajah, Janarthanan; Hamilton, Andrew D.; Brodbelt, Jennifer S.

2010-01-01

Isoguanine (2-oxo-6-amino-guanine), a natural but non-standard base, exhibits unique self-association properties compared to its isomer, guanine, and results in formation of different higher order DNA structures. In this work, the higher order structures formed by oligonucleotides containing guanine repeats or isoguanine repeats after annealing in solutions containing various cations are evaluated by electrospray ionization mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy. The guanine-containing strand (G9) consistently formed quadruplexes upon annealing, whereas the isoguanine strand (Ig9) formed both pentaplexes and quadruplexes depending on the annealing cation. Quadruplex formation with G9 showed some dependence on the identity of the cation present during annealing with high relative quadruplex formation detected with six of ten cations. Analogous annealing experiments with Ig9 resulted in complex formation with all ten cations, and the majority of the resulting complexes were pentaplexes. CD results indicated most of the original complexes survived the desalting process necessary for ESI-MS analysis. In addition, several complexes, especially the pentaplexes, were found to be capable of cation exchange with ammonium ions. Ab initio calculations were conducted for isoguanine tetrads and pentads coordinated with all ten cations to predict the most energetically stable structures of the complexes in the gas phase. The observed preference of forming quadruplexes versus pentaplexes as a function of the coordinated cation can be interpreted by the calculated reaction energies of both the tetrads and pentads in combination with the distortion energies of tetrads. PMID:19746468

A Method for WD40 Repeat Detection and Secondary Structure Prediction

PubMed Central

Wang, Yang; Jiang, Fan; Zhuo, Zhu; Wu, Xian-Hui; Wu, Yun-Dong

2013-01-01

WD40-repeat proteins (WD40s), as one of the largest protein families in eukaryotes, play vital roles in assembling protein-protein/DNA/RNA complexes. WD40s fold into similar β-propeller structures despite diversified sequences. A program WDSP (WD40 repeat protein Structure Predictor) has been developed to accurately identify WD40 repeats and predict their secondary structures. The method is designed specifically for WD40 proteins by incorporating both local residue information and non-local family-specific structural features. It overcomes the problem of highly diversified protein sequences and variable loops. In addition, WDSP achieves a better prediction in identifying multiple WD40-domain proteins by taking the global combination of repeats into consideration. In secondary structure prediction, the average Q3 accuracy of WDSP in jack-knife test reaches 93.7%. A disease related protein LRRK2 was used as a representive example to demonstrate the structure prediction. PMID:23776530

Translation of dipeptide repeat proteins from the C9ORF72 expanded repeat is associated with cellular stress.

PubMed

Sonobe, Yoshifumi; Ghadge, Ghanashyam; Masaki, Katsuhisa; Sendoel, Ataman; Fuchs, Elaine; Roos, Raymond P

2018-08-01

Expansion of a hexanucleotide repeat (HRE), GGGGCC, in the C9ORF72 gene is recognized as the most common cause of familial amyotrophic lateral sclerosis (FALS), frontotemporal dementia (FTD) and ALS-FTD, as well as 5-10% of sporadic ALS. Despite the location of the HRE in the non-coding region (with respect to the main C9ORF72 gene product), dipeptide repeat proteins (DPRs) that are thought to be toxic are translated from the HRE in all three reading frames from both the sense and antisense transcript. Here, we identified a CUG that has a good Kozak consensus sequence as the translation initiation codon. Mutation of this CTG significantly suppressed polyglycine-alanine (GA) translation. GA was translated when the G 4 C 2 construct was placed as the second cistron in a bicistronic construct. CRISPR/Cas9-induced knockout of a non-canonical translation initiation factor, eIF2A, impaired GA translation. Transfection of G 4 C 2 constructs induced an integrated stress response (ISR), while triggering the ISR led to a continuation of translation of GA with a decline in conventional cap-dependent translation. These in vitro observations were confirmed in chick embryo neural cells. The findings suggest that DPRs translated from an HRE in C9ORF72 aggregate and lead to an ISR that then leads to continuing DPR production and aggregation, thereby creating a continuing pathogenic cycle. Copyright © 2018 Elsevier Inc. All rights reserved.

Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

PubMed Central

Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

1995-01-01

The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF. Images PMID:7501455

Chicken Interferon-induced Protein with Tetratricopeptide Repeats 5 Antagonizes Replication of RNA Viruses.

PubMed

Santhakumar, Diwakar; Rohaim, Mohammed Abdel Mohsen Shahaat; Hussein, Hussein A; Hawes, Pippa; Ferreira, Helena Lage; Behboudi, Shahriar; Iqbal, Munir; Nair, Venugopal; Arns, Clarice W; Munir, Muhammad

2018-05-01

The intracellular actions of interferon (IFN)-regulated proteins, including IFN-induced proteins with tetratricopeptide repeats (IFITs), attribute a major component of the protective antiviral host defense. Here we applied genomics approaches to annotate the chicken IFIT locus and currently identified a single IFIT (chIFIT5) gene. The profound transcriptional level of this effector of innate immunity was mapped within its unique cis-acting elements. This highly virus- and IFN-responsive chIFIT5 protein interacted with negative sense viral RNA structures that carried a triphosphate group on its 5' terminus (ppp-RNA). This interaction reduced the replication of RNA viruses in lentivirus-mediated IFIT5-stable chicken fibroblasts whereas CRISPR/Cas9-edited chIFIT5 gene knockout fibroblasts supported the replication of RNA viruses. Finally, we generated mosaic transgenic chicken embryos stably expressing chIFIT5 protein or knocked-down for endogenous chIFIT5 gene. Replication kinetics of RNA viruses in these transgenic chicken embryos demonstrated the antiviral potential of chIFIT5 in ovo. Taken together, these findings propose that IFIT5 specifically antagonize RNA viruses by sequestering viral nucleic acids in chickens, which are unique in innate immune sensing and responses to viruses of both poultry and human health significance.

High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system.

PubMed

Yosef, Ido; Goren, Moran G; Kiro, Ruth; Edgar, Rotem; Qimron, Udi

2011-12-13

Prokaryotic DNA arrays arranged as clustered regularly interspaced short palindromic repeats (CRISPR), along with their associated proteins, provide prokaryotes with adaptive immunity by RNA-mediated targeting of alien DNA or RNA matching the sequences between the repeats. Here, we present a thorough screening system for the identification of bacterial proteins participating in immunity conferred by the Escherichia coli CRISPR system. We describe the identification of one such protein, high-temperature protein G (HtpG), a homolog of the eukaryotic chaperone heat-shock protein 90. We demonstrate that in the absence of htpG, the E. coli CRISPR system loses its suicidal activity against λ prophage and its ability to provide immunity from lysogenization. Transcomplementation of htpG restores CRISPR activity. We further show that inactivity of the CRISPR system attributable to htpG deficiency can be suppressed by expression of Cas3, a protein that is essential for its activity. Accordingly, we also find that the steady-state level of overexpressed Cas3 is significantly enhanced following HtpG expression. We conclude that HtpG is a newly identified positive modulator of the CRISPR system that is essential for maintaining functional levels of Cas3.

High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system

PubMed Central

Yosef, Ido; Goren, Moran G.; Kiro, Ruth; Edgar, Rotem; Qimron, Udi

2011-01-01

Prokaryotic DNA arrays arranged as clustered regularly interspaced short palindromic repeats (CRISPR), along with their associated proteins, provide prokaryotes with adaptive immunity by RNA-mediated targeting of alien DNA or RNA matching the sequences between the repeats. Here, we present a thorough screening system for the identification of bacterial proteins participating in immunity conferred by the Escherichia coli CRISPR system. We describe the identification of one such protein, high-temperature protein G (HtpG), a homolog of the eukaryotic chaperone heat-shock protein 90. We demonstrate that in the absence of htpG, the E. coli CRISPR system loses its suicidal activity against λ prophage and its ability to provide immunity from lysogenization. Transcomplementation of htpG restores CRISPR activity. We further show that inactivity of the CRISPR system attributable to htpG deficiency can be suppressed by expression of Cas3, a protein that is essential for its activity. Accordingly, we also find that the steady-state level of overexpressed Cas3 is significantly enhanced following HtpG expression. We conclude that HtpG is a newly identified positive modulator of the CRISPR system that is essential for maintaining functional levels of Cas3. PMID:22114197

Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity.

PubMed

Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong

2011-09-02

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca(2+) was located at strategic positions in the oligomerization interface. We further showed that removal of Ca(2+) ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca(2+) ions.

Up-regulation of Hyperpolarization-activated Cyclic Nucleotide-gated Channel 3 (HCN3) by Specific Interaction with K+ Channel Tetramerization Domain-containing Protein 3 (KCTD3)*

PubMed Central

Cao-Ehlker, Xiaochun; Zong, Xiangang; Hammelmann, Verena; Gruner, Christian; Fenske, Stefanie; Michalakis, Stylianos; Wahl-Schott, Christian; Biel, Martin

2013-01-01

Most ion channels consist of the principal ion-permeating core subunit(s) and accessory proteins that are assembled with the channel core. The biological functions of the latter proteins are diverse and include the regulation of the biophysical properties of the ion channel, its connection to signaling pathways and the control of its cell surface expression. There is recent evidence that native hyperpolarization-activated cyclic nucleotide-gated channel complexes (HCN1–4) also contain accessory subunits, among which TRIP8b (tetratricopeptide repeat-containing Rab8b-interacting protein) has been most extensively studied. Here, we identify KCTD3, a so far uncharacterized member of the potassium channel tetramerization-domain containing (KCTD) protein family as an HCN3-interacting protein. KCTD3 is widely expressed in brain and some non-neuronal tissues and colocalizes with HCN3 in specific regions of the brain including hypothalamus. Within the HCN channel family, KCTD3 specifically binds to HCN3 and leads to a profound up-regulation of cell surface expression and current density of this channel. HCN3 can also functionally interact with TRIP8b; however, we found no evidence for channel complexes containing both TRIP8b and KCTD3. The C terminus of HCN3 is crucially required for functional interaction with KCTD3. Replacement of the cytosolic C terminus of HCN2 by the corresponding domain of HCN3 renders HCN2 sensitive to regulation by KCTD3. The C-terminal-half of KCTD3 is sufficient for binding to HCN3. However, the complete protein including the N-terminal tetramerization domain is needed for HCN3 current up-regulation. Together, our experiments indicate that KCTD3 is an accessory subunit of native HCN3 complexes. PMID:23382386

Requirement of the Cytosolic Interaction between PATHOGENESIS-RELATED PROTEIN10 and LEUCINE-RICH REPEAT PROTEIN1 for Cell Death and Defense Signaling in Pepper[W

PubMed Central

Choi, Du Seok; Hwang, In Sun; Hwang, Byung Kook

2012-01-01

Plants recruit innate immune receptors such as leucine-rich repeat (LRR) proteins to recognize pathogen attack and activate defense genes. Here, we identified the pepper (Capsicum annuum) pathogenesis-related protein10 (PR10) as a leucine-rich repeat protein1 (LRR1)–interacting partner. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirmed the specific interaction between LRR1 and PR10 in planta. Avirulent Xanthomonas campestris pv vesicatoria infection induces PR10 expression associated with the hypersensitive cell death response. Transient expression of PR10 triggers hypersensitive cell death in pepper and Nicotiana benthamiana leaves, which is amplified by LRR1 coexpression as a positive regulator. LRR1 promotes the ribonuclease activity and phosphorylation of PR10, leading to enhanced cell death signaling. The LRR1-PR10 complex is formed in the cytoplasm, resulting in its secretion into the apoplastic space. Engineered nuclear confinement of both proteins revealed that the cytoplasmic localization of the PR10-LRR1 complex is essential for cell death–mediated defense signaling. PR10/LRR1 silencing in pepper compromises resistance to avirulent X. campestris pv vesicatoria infection. By contrast, PR10/LRR1 overexpression in Arabidopsis thaliana confers enhanced resistance to Pseudomonas syringae pv tomato and Hyaloperonospora arabidopsidis. Together, these results suggest that the cytosolic LRR-PR10 complex is responsible for cell death–mediated defense signaling. PMID:22492811

Exaggerated phosphorylation of brain tau protein in CRH KO mice exposed to repeated immobilization stress.

PubMed

Kvetnansky, Richard; Novak, Petr; Vargovic, Peter; Lejavova, Katarina; Horvathova, Lubica; Ondicova, Katarina; Manz, George; Filipcik, Peter; Novak, Michal; Mravec, Boris

2016-07-01

Neuroendocrine and behavioral stress responses are orchestrated by corticotropin-releasing hormone (CRH) and norepinephrine (NE) synthesizing neurons. Recent findings indicate that stress may promote development of neurofibrillary pathology in Alzheimer's disease. Therefore, we investigated relationships among stress, tau protein phosphorylation, and brain NE using wild-type (WT) and CRH-knockout (CRH KO) mice. We assessed expression of phosphorylated tau (p-tau) at the PHF-1 epitope and NE concentrations in the locus coeruleus (LC), A1/C1 and A2/C2 catecholaminergic cell groups, hippocampus, amygdala, nucleus basalis magnocellularis, and frontal cortex of unstressed, singly stressed or repeatedly stressed mice. Moreover, gene expression and protein levels of tyrosine hydroxylase (TH) and CRH receptor mRNA were determined in the LC. Plasma corticosterone levels were also measured. Exposure to a single stress increases tau phosphorylation throughout the brain in WT mice when compared to singly stressed CRH KO animals. In contrast, repeatedly stressed CRH KO mice showed exaggerated tau phosphorylation relative to WT controls. We also observed differences in extent of tau phosphorylation between investigated structures, e.g. the LC and hippocampus. Moreover, CRH deficiency leads to different responses to stress in gene expression of TH, NE concentrations, CRH receptor mRNA, and plasma corticosterone levels. Our data indicate that CRH effects on tau phosphorylation are dependent on whether stress is single or repeated, and differs between brain regions. Our findings indicate that CRH attenuates mechanisms responsible for development of stress-induced tau neuropathology, particularly in conditions of chronic stress. However, the involvement of central catecholaminergic neurons in these mechanisms remains unclear and is in need of further investigation.

The effect of a diet containing 70% protein from plants on mineral metabolism and musculoskeletal health in chronic kidney disease.

PubMed

Moorthi, Ranjani N; Armstrong, Cheryl L H; Janda, Kevin; Ponsler-Sipes, Kristen; Asplin, John R; Moe, Sharon M

2014-01-01

Chronic Kidney Disease (CKD) is associated with alterations in phosphorus excretion, and increases in fibroblast growth factor (FGF23) and parathyroid hormone (PTH). Plant protein-based phytate-bound phosphorus, is less bioavailable than that from animal sources. Our one-week study that was conducted previously showed that a nearly 100% plant protein-based diet benefits mineral metabolism in CKD; however, this diet may not be acceptable to patients. Here we hypothesize that a diet containing 70% protein from plants has similar efficacy and is tolerated by CKD patients. Thirteen subjects with CKD 3-4 received an omnivorous diet containing 70% protein from plants for 4 weeks. The primary outcome was change in 24 h urine phosphorus. Secondary outcomes were changes in serum phosphorus, FGF23, PTH, urine sodium excretion, grip strength and fat free mass. Repeated measures analysis of variance (ANOVA) was used to test differences in parameters over the 4 weeks. Mean age of subjects was 54.8 years. Median eGFR was 26 (IQR 14.7) ml/min/1.73 m(2). Over the 4-week period, urine phosphorus significantly decreased by 215 ± 232 mg/day (p < 0.001). No significant changes in serum FGF23, phosphorus or PTH were noted. Urine sodium and titratable acid decreased significantly on the diet. Hand grip strength and fat-free mass did not change. There were two hyperkalemia events both 5.8 mEq/l, corrected by food substitutions. No other adverse events were observed. A 70% plant protein diet is safe, tolerated, and efficacious in lowering urine phosphorus excretion and may be an alternative to phosphate binders. © 2015 S. Karger AG, Basel.

The Effect of a Diet containing 70% Protein from Plants on Mineral Metabolism and Musculoskeletal Health in Chronic Kidney Disease

PubMed Central

Moorthi, Ranjani N; Armstrong, Cheryl L. H.; Janda, Kevin; Ponsler-Sipes, Kristen; Asplin, John R.; Moe, Sharon M

2015-01-01

Background Chronic Kidney Disease (CKD) is associated with alterations in phosphorus excretion, and increases in fibroblast growth factor (FGF23) and parathyroid hormone (PTH). Plant protein based phytate bound phosphorus, is less bioavailable than that from animal sources. Our prior one week study showed that a nearly 100% plant protein based diet benefits mineral metabolism in CKD; however this diet may not be acceptable to patients. Here we hypothesize that a diet containing 70% protein from plants has similar efficacy and is tolerated by CKD patients. Methods Thirteen subjects with CKD 3-4 received an omnivore diet containing 70% protein from plants for 4 weeks. The primary outcome was change in 24 hour urine phosphorus. Secondary outcomes were changes in serum phosphorus, FGF23, PTH, urine sodium excretion, grip strength and fat free mass. Repeated measures analysis of variance (ANOVA) was used to test differences in parameters over the 4 weeks. Results Mean age of subjects was 54.8 years. Median eGFR was 26 (IQR 14.7) ml/min/1.73m2. Over the 4 week period, urine phosphorus significantly decreased by 215±232 mg/day (p<0.001). No significant changes in serum FGF23, phosphorus or PTH were noted. Urine sodium and titratable acid decreased significantly on the diet. Hand grip strength and fat-free mass did not change. There were two hyperkalemia events both 5.8 meq/l, corrected by food substitutions. No other adverse events were observed. Conclusions A 70% plant protein diet is safe, tolerated, and efficacious in lowering urine phosphorus excretion and may be an alternative to phosphate binders. PMID:25613675

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence.

PubMed

Lahr, Roni M; Mack, Seshat M; Héroux, Annie; Blagden, Sarah P; Bousquet-Antonelli, Cécile; Deragon, Jean-Marc; Berman, Andrea J

2015-09-18

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence

DOE PAGES

Lahr, Roni M.; Mack, Seshat M.; Heroux, Annie; ...

2015-07-22

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. Amore » putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. Ultimately, these studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.« less

A Conserved Region between the Heptad Repeats of Paramyxovirus Fusion Proteins is Critical for Proper F Protein Folding†

PubMed Central

Gardner, Amanda E.; Martin, Kimberly L.; Dutch, Rebecca E.

2008-01-01

Paramyxoviruses are a diverse family which utilizes a fusion (F) protein to enter cells via fusion of the viral lipid bilayer with a target cell membrane. Although certain regions of F are known to play critical roles in membrane fusion, the function of much of the protein remains unclear. Sequence alignment of a set of paramyxovirus F proteins and analysis utilizing Block Maker identified a region of conserved amino acid sequence in a large domain between the heptad repeats of F1, designated CBF1. We employed site-directed mutagenesis to analyze the function of completely conserved residues of CBF1 in both the simian virus 5 (SV5) and Hendra virus F proteins. The majority of CBF1 point mutants were deficient in homotrimer formation, proteolytic processing, and transport to the cell surface. For some SV5 F mutants, proteolytic cleavage and surface expression could be restored by expression at 30°C, and varying levels of fusion promotion were observed at this temperature. In addition, the mutant SV5 F V402A displayed a hyperfusogenic phenotype at both 30°C and 37°C, indicating this mutation allows for efficient fusion with only an extremely small amount of cleaved, active protein. The recently published prefusogenic structure of PIV5/SV5 F [Yin, H.S., et al. (2006) Nature 439, 38–44] indicates that residues within and flanking CBF1 interact with the fusion peptide domain. Together, these data suggest that CBF1-fusion peptide interactions are critical for the initial folding of paramyxovirus F proteins from across this important viral family, and can also modulate subsequent membrane fusion promotion. PMID:17417875

Repeat exposure to group A streptococcal M protein exacerbates cardiac damage in a rat model of rheumatic heart disease.

PubMed

Gorton, Davina; Sikder, Suchandan; Williams, Natasha L; Chilton, Lisa; Rush, Catherine M; Govan, Brenda L; Cunningham, Madeleine W; Ketheesan, Natkunam

2016-12-01

Rheumatic fever and rheumatic heart disease (RF/RHD) develop following repeated infection with group A streptococci (GAS). We used the Rat Autoimmune Valvulitis (RAV) model of RF/RHD to demonstrate that repetitive booster immunization with GAS-derived recombinant M protein (rM5) resulted in an enhanced anti-cardiac myosin antibody response that may contribute to the breaking of immune tolerance leading to RF/RHD and increased infiltration of heart valves by mononuclear cells. With each boost, more inflammatory cells were observed infiltrating heart tissue which could lead to severe cardiac damage. We also found evidence that both complement and anti-M protein antibodies in serum from rM5-immunized rats have the potential to contribute to inflammation in heart valves by activating cardiac endothelium. More importantly, we have demonstrated by electrocardiography for the first time in the RAV model that elongation of P-R interval follows repetitive boost with rM5. Our observations provide experimental evidence for cardiac alterations following repeated exposure to GAS M protein with immunological and electrophysiological features resembling that seen in humans following recurrent GAS infection.

Impact assessment of projected climate change on diffuse phosphorous loss in Xin'anjiang catchment, China.

PubMed

Zhai, Xiaoyan; Zhang, Yongyong

2018-02-01

Diffuse nutrient loss is a serious threat to water security and has severely deteriorated water quality throughout the world. Xin'anjiang catchment, as a main drinking water source for Hangzhou City, has been a national concern for water environment protection with payment for watershed services construction. Detection of diffuse phosphorous (DP) pollution dynamics under climate change is significant for sustainable water quality management. In this study, the impact of projected climate change on DP load was analyzed using SWAT to simulate the future changes of diffuse components (carriers: water discharge and sediment; nutrient: DP) at both station and sub-catchment scales under three climate change scenarios (RCP2.6, RCP4.5, and RCP8.5). Results showed that wetting and warming years were expected with increasing tendencies of both precipitation and temperature in the two future periods (2020s: 2021~2030, 2030s: 2031~2040) except in the 2020s in the RCP2.6 scenario, and the annual average increasing ratios of precipitation and temperature reached - 1.79~3.79% and 0.48~1.27 °C, respectively, comparing with those in the baseline (2000s: 2001~2010). Climate change evidently altered annual and monthly average water discharge and sediment load, while it has a remarkable impact on the timing and monthly value of DP load at station scale. DP load tended to increase in the non-flood season at Yuliang due to strengthened nutrient flushing from rice land into rivers with increasing precipitation and enhanced phosphorous cycle in soil layers with increasing temperature, while it tended to decrease in the flood season at Yuliang and in most months at Tunxi due to restricted phosphorous reaction with reduced dissolved oxygen content and enhanced dilution effect. Spatial variability existed in the changes of sediment load and DP load at sub-catchment scale due to climate change. DP load tended to decrease in most sub-catchments and was the most remarkable in the RCP8

Reduced C9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the C9ORF72 hexanucleotide repeat expansion.

PubMed

Waite, Adrian J; Bäumer, Dirk; East, Simon; Neal, James; Morris, Huw R; Ansorge, Olaf; Blake, Derek J

2014-07-01

An intronic G(4)C(2) hexanucleotide repeat expansion in C9ORF72 is a major cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Several mechanisms including RNA toxicity, repeat-associated non-AUG translation mediated dipeptide protein aggregates, and haploinsufficiency of C9orf72 have been implicated in the molecular pathogenesis of this disorder. The aims of this study were to compare the use of two different Southern blot probes for detection of repeat expansions in an amyotrophic lateral sclerosis and frontotemporal lobar degeneration pathological cohort and to determine the levels of C9orf72 transcript variants and protein isoforms in patients versus control subjects. Our Southern blot studies identified smaller repeat expansions (250-1800 bp) that were only detectable with the flanking probe highlighting the potential for divergent results using different Southern blotting protocols that could complicate genotype-phenotype correlation studies. Further, we characterize a new C9orf72 antibody and show for the first time decreased C9orf72 protein levels in the frontal cortex from patients with a pathological hexanucleotide repeat expansion. These data suggest that a reduction in C9orf72 protein may be a consequence of the disease. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

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

Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy

2010-10-22

Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated withmore » reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.« less

Positive selection and propeptide repeats promote rapid interspecific divergence of a gastropod sperm protein.

PubMed

Hellberg, M E; Moy, G W; Vacquier, V D

2000-03-01

Male-specific proteins have increasingly been reported as targets of positive selection and are of special interest because of the role they may play in the evolution of reproductive isolation. We report the rapid interspecific divergence of cDNA encoding a major acrosomal protein of unknown function (TMAP) of sperm from five species of teguline gastropods. A mitochondrial DNA clock (calibrated by congeneric species divided by the Isthmus of Panama) estimates that these five species diverged 2-10 MYA. Inferred amino acid sequences reveal a propeptide that has diverged rapidly between species. The mature protein has diverged faster still due to high nonsynonymous substitution rates (> 25 nonsynonymous substitutions per site per 10(9) years). cDNA encoding the mature protein (89-100 residues) shows evidence of positive selection (Dn/Ds > 1) for 4 of 10 pairwise species comparisons. cDNA and predicted secondary-structure comparisons suggest that TMAP is neither orthologous nor paralogous to abalone lysin, and thus marks a second, phylogenetically independent, protein subject to strong positive selection in free-spawning marine gastropods. In addition, an internal repeat in one species (Tegula aureotincta) produces a duplicated cleavage site which results in two alternatively processed mature proteins differing by nine amino acid residues. Such alternative processing may provide a mechanism for introducing novel amino acid sequence variation at the amino-termini of proteins. Highly divergent TMAP N-termini from two other tegulines (Tegula regina and Norrisia norrisii) may have originated by such a mechanism.

Repeat-Associated Plasticity in the Helicobacter pylori RD Gene Family▿ †

PubMed Central

Shak, Joshua R.; Dick, Jonathan J.; Meinersmann, Richard J.; Perez-Perez, Guillermo I.; Blaser, Martin J.

2009-01-01

The bacterium Helicobacter pylori is remarkable for its ability to persist in the human stomach for decades without provoking sterilizing immunity. Since repetitive DNA can facilitate adaptive genomic flexibility via increased recombination, insertion, and deletion, we searched the genomes of two H. pylori strains for nucleotide repeats. We discovered a family of genes with extensive repetitive DNA that we have termed the H. pylori RD gene family. Each gene of this family is composed of a conserved 3′ region, a variable mid-region encoding 7 and 11 amino acid repeats, and a 5′ region containing one of two possible alleles. Analysis of five complete genome sequences and PCR genotyping of 42 H. pylori strains revealed extensive variation between strains in the number, location, and arrangement of RD genes. Furthermore, examination of multiple strains isolated from a single subject's stomach revealed intrahost variation in repeat number and composition. Despite prior evidence that the protein products of this gene family are expressed at the bacterial cell surface, enzyme-linked immunosorbent assay and immunoblot studies revealed no consistent seroreactivity to a recombinant RD protein by H. pylori-positive hosts. The pattern of repeats uncovered in the RD gene family appears to reflect slipped-strand mispairing or domain duplication, allowing for redundancy and subsequent diversity in genotype and phenotype. This novel family of hypervariable genes with conserved, repetitive, and allelic domains may represent an important locus for understanding H. pylori persistence in its natural host. PMID:19749042

A dominant sulfhydryl-containing protein in the outer membrane of Neisseria gonorrhoeae.

PubMed Central

Norrod, E P; Browne, S L; Feldweg, A; Leonard, J

1993-01-01

By using a method that labels sulfhydryl-containing proteins in situ, we have detected a major outer membrane protein of Neisseria gonorrhoeae at 41 kDa. A protein of this molecular mass has not previously been shown to be a major outer membrane protein in gonococci. In addition, a minor protein rich in cysteinyl residues was detected at 31.5 kDa. Images PMID:8432710

Collagen triple helix repeat containing 1 is a new promigratory marker of arthritic pannus.

PubMed

Shekhani, Mohammed Talha; Forde, Toni S; Adilbayeva, Altynai; Ramez, Mohamed; Myngbay, Askhat; Bexeitov, Yergali; Lindner, Volkhard; Adarichev, Vyacheslav A

2016-07-19

The formation of destructive hypercellular pannus is critical to joint damage in rheumatoid arthritis (RA). The collagen triple helix repeat containing 1 (CTHRC1) protein expressed by activated stromal cells of diverse origin has previously been implicated in tissue remodeling and carcinogenesis. We recently discovered that the synovial Cthrc1 mRNA directly correlates with arthritis severity in mice. This study characterizes the role of CTHRC1 in arthritic pannus formation. Synovial joints of mice with collagen antibody-induced arthritis (CAIA) and human RA-fibroblast-like synoviocytes (FLS) were immunostained for CTHRC1, FLS and macrophage-specific markers. CTHRC1 levels in plasma from patients with RA were measured using sandwich ELISA. The migratory response of fibroblasts was studied with a transwell migration assay and time-lapse microscopy. Velocity and directness of cell migration was analyzed by recording the trajectories of cells treated with rhCTHRC1. Immunohistochemical analysis of normal and inflamed synovium revealed highly inducible expression of CTHRC1 in arthritis (10.9-fold). At the tissue level, CTHRC1-expressing cells occupied the same niche as large fibroblast-like cells positive for α-smooth muscle actin (α-SMA) and cadherin 11 (CDH11). CTHRC1 was produced by activated FLS predominantly located at the synovial intimal lining and at the bone-pannus interface. Cultured RA-FLS expressed CDH11, α-SMA, and CTHRC1. Upon treatment with exogenous rhCTHRC1, embryonic fibroblasts and RA-FLS significantly increased migration velocity, directness, and cell length along the front-tail axis (1.4-fold, p < 0.01). CTHRC1 was established as a novel marker of activated synoviocytes in murine experimental arthritis and RA. The pro-migratory effect of CTHRC1 on synoviocytes is considered one of the mechanisms promoting hypercellularity of the arthritic pannus.

Impact of the excision of an ancient repeat insertion on Rickettsia conorii guanylate kinase activity.

PubMed

Abergel, Chantal; Blanc, Guillaume; Monchois, Vincent; Renesto, Patricia; Sigoillot, Cécile; Ogata, Hiroyuki; Raoult, Didier; Claverie, Jean-Michel

2006-11-01

The genomic sequencing of Rickettsia conorii revealed a new family of Rickettsia-specific palindromic elements (RPEs) capable of in-frame insertion in preexisting open reading frames (ORFs). Many of these altered ORFs correspond to proteins with well-characterized or essential functions in other microorganisms. Previous experiments indicated that RPE-containing genes are normally transcribed and that no excision of the repeat occurs at the mRNA level. Using mass spectrometry, we now confirmed the retention of the RPE-derived amino acid residues in 4 proteins successfully expressed in Escherichia coli, raising the general question of the consequences of this common insertion event on the fitness of Rickettsia enzymes. The predicted guanylate kinase activity of the R. conorii gmk gene product was measured both on the RPE-containing and RPE-excised recombinant proteins. We show that the 2 proteins are active but exhibit substantial differences in their affinity for adenosine triphosphate, guanosine monophosphate, and catalytic constants. The distribution of the RPEgmk insert among Rickettsia species indicates that the insertion event is ancient and occurred after the divergence of Rickettsia felis and R. conorii but before that of Rickettsia helvetica and R. conorii. We found no evidence that the gmk gene fixed adaptive changes to compensate the RPE peptide insertion. Furthermore, the analysis of the rates of divergence in 23 RPE-containing genes indicates that coding RPE repeats tend to evolve under weak selective constraint, at a rate similar to intergenic noncoding RPE sequences. Altogether, these results suggest that the insertion of RPE-encoded "selfish peptides," although respecting the original fold and activity of the host proteins, might be slightly detrimental to the enzyme efficiency within limits tolerable for slow-growing intracellular parasites such as Rickettsia.

A Hot-Spot Motif Characterizes the Interface between a Designed Ankyrin-Repeat Protein and Its Target Ligand

PubMed Central

Cheung, Luthur Siu-Lun; Kanwar, Manu; Ostermeier, Marc; Konstantopoulos, Konstantinos

2012-01-01

Nonantibody scaffolds such as designed ankyrin repeat proteins (DARPins) can be rapidly engineered to detect diverse target proteins with high specificity and offer an attractive alternative to antibodies. Using molecular simulations, we predicted that the binding interface between DARPin off7 and its ligand (maltose binding protein; MBP) is characterized by a hot-spot motif in which binding energy is largely concentrated on a few amino acids. To experimentally test this prediction, we fused MBP to a transmembrane domain to properly orient the protein into a polymer-cushioned lipid bilayer, and characterized its interaction with off7 using force spectroscopy. Using this, to our knowledge, novel technique along with surface plasmon resonance, we validated the simulation predictions and characterized the effects of select mutations on the kinetics of the off7-MBP interaction. Our integrated approach offers scientific insights on how the engineered protein interacts with the target molecule. PMID:22325262

Lipid transfer proteins in the assembly of apoB-containing lipoproteins.

PubMed

Sirwi, Alaa; Hussain, M Mahmood

2018-04-12

A better understanding of intracellular lipoprotein assembly may help identify proteins with important roles in lipid disorders. ApoB-containing lipoproteins are macromolecular lipid and protein micelles that act as specialized transport vehicles for hydrophobic lipids. They are assembled predominantly in enterocytes and hepatocytes to transport dietary and endogenous fat, respectively, to different tissues. Assembly occurs in the endoplasmic reticulum and is dependent on lipid re-synthesis in the endoplasmic reticulum and on a chaperone, namely microsomal triglyceride transfer protein. Precursors for lipid synthesis are obtained from extracellular sources and from cytoplasmic lipid droplets. Microsomal triglyceride transfer protein is the major and essential lipid transfer protein that transfers phospholipids and triacylglycerols to nascent apoB for the assembly of lipoproteins. Assembly is aided by cell death-inducing DFF45-like effector B and by phospholipid transfer protein, which may facilitate additional deposition of triacylglycerols and phospholipids, respectively, to apoB. Here, we summarize the current understanding of the different steps in the assembly of apoB-containing lipoproteins and discuss the role of lipid transfer proteins in these steps to help identify new clinical targets for lipid-associated disorders, such as heart disease. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Staphylococcus aureus activates type I IFN signaling in mice and humans through the Xr repeated sequences of protein A

PubMed Central

Martin, Francis J.; Gomez, Marisa I.; Wetzel, Dawn M.; Memmi, Guido; O’Seaghdha, Maghnus; Soong, Grace; Schindler, Christian; Prince, Alice

2009-01-01

The activation of type I IFN signaling is a major component of host defense against viral infection, but it is not typically associated with immune responses to extracellular bacterial pathogens. Using mouse and human airway epithelial cells, we have demonstrated that Staphylococcus aureus activates type I IFN signaling, which contributes to its virulence as a respiratory pathogen. This response was dependent on the expression of protein A and, more specifically, the Xr domain, a short sequence–repeat region encoded by DNA that consists of repeated 24-bp sequences that are the basis of an internationally used epidemiological typing scheme. Protein A was endocytosed by airway epithelial cells and subsequently induced IFN-β expression, JAK-STAT signaling, and IL-6 production. Mice lacking IFN-α/β receptor 1 (IFNAR-deficient mice), which are incapable of responding to type I IFNs, were substantially protected against lethal S. aureus pneumonia compared with wild-type control mice. The profound immunological consequences of IFN-β signaling, particularly in the lung, may help to explain the conservation of multiple copies of the Xr domain of protein A in S. aureus strains and the importance of protein A as a virulence factor in the pathogenesis of staphylococcal pneumonia. PMID:19603548

Analysis of polyglutamine-coding repeats in the TATA-binding protein in different human populations and in patients with schizophrenia an bipolar affective disorder

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

Rubinsztein, D.C.; Leggo, J.; Crow, T.J.

A new class of disease (including Huntington disease, Kennedy disease, and spinocerebellar ataxias types 1 and 3) results from abnormal expansions of CAG trinucleotides in the coding regions of genes. In all of these diseases the CAG repeats are thought to be translated into polyglutamine tracts. There is accumulating evidence arguing for CAG trinucleotide expansions as one of the causative disease mutations in schizophrenia and bipolar affective disorder. We and others believe that the TATA-binding protein (TBP) is an important candidate to investigate in these diseases as it contains a highly polymorphic stretch of glutamine codons, which are close tomore » the threshold length where the polyglutamine tracts start to be associated with disease. Thus, we examined the lengths of this polyglutamine repeat in normal unrelated East Anglians, South African Blacks, sub-Saharan Africans mainly from Nigeria, and Asian Indians. We also examined 43 bipolar affective disorder patients and 65 schizophrenic patients. The range of polyglutamine tract-lengths that we found in humans was from 26-42 codons. No patients with bipolar affective disorder and schizophrenia had abnormal expansions at this locus. 22 refs., 1 tab.« less

Repeated Exposure to D-Amphetamine Decreases Global Protein Synthesis and Regulates the Translation of a Subset of mRNAs in the Striatum

PubMed Central

Biever, Anne; Boubaker-Vitre, Jihane; Cutando, Laura; Gracia-Rubio, Irene; Costa-Mattioli, Mauro; Puighermanal, Emma; Valjent, Emmanuel

2017-01-01

Repeated psychostimulant exposure induces persistent gene expression modifications that contribute to enduring changes in striatal GABAergic spiny projecting neurons (SPNs). However, it remains unclear whether changes in the control of mRNA translation are required for the establishment of these durable modifications. Here we report that repeated exposure to D-amphetamine decreases global striatal mRNA translation. This effect is paralleled by an enhanced phosphorylation of the translation factors, eIF2α and eEF2, and by the concomitant increased translation of a subset of mRNAs, among which the mRNA encoding for the activity regulated cytoskeleton-associated protein, also known as activity regulated gene 3.1 (Arc/Arg3.1). The enrichment of Arc/Arg3.1 mRNA in the polysomal fraction is accompanied by a robust increase of Arc/Arg3.1 protein levels within the striatum. Immunofluorescence analysis revealed that this increase occurred preferentially in D1R-expressing SPNs localized in striosome compartments. Our results suggest that the decreased global protein synthesis following repeated exposure to D-amphetamine favors the translation of a specific subset of mRNAs in the striatum. PMID:28119566

Stabilization of perfect and imperfect tandem repeats by single-strand DNA exonucleases

PubMed Central

Feschenko, Vladimir V.; Rajman, Luis A.; Lovett, Susan T.

2003-01-01

Rearrangements between tandemly repeated DNA sequences are a common source of genetic instability. Such rearrangements underlie several human genetic diseases. In many organisms, the mismatch-repair (MMR) system functions to stabilize repeats when the repeat unit is short or when sequence imperfections are present between the repeats. We show here that the action of single-stranded DNA (ssDNA) exonucleases plays an additional, important role in stabilizing tandem repeats, independent of their role in MMR. For perfect repeats of ≈100 bp in Escherichia coli that are not susceptible to MMR, exonuclease (Exo)-I, ExoX, and RecJ exonuclease redundantly inhibit deletion. Our data suggest that >90% of potential deletion events are avoided by the combined action of these three exonucleases. Imperfect tandem repeats, less prone to rearrangements, are stabilized by both the MMR-pathway and ssDNA-specific exonucleases. For 100-bp repeats containing four mispairs, ExoI alone aborts most deletion events, even in the presence of a functional MMR system. By genetic analysis, we show that the inhibitory effect of ssDNA exonucleases on deletion formation is independent of the MutS and UvrD proteins. Exonuclease degradation of DNA displaced during the deletion process may abort slipped misalignment. Exonuclease action is therefore a significant force in genetic stabilization of many forms of repetitive DNA. PMID:12538867

Characterization of a double WAP domain-containing protein from the red swamp crayfish Procambarus clarkii

USDA-ARS?s Scientific Manuscript database

Crustaceans express multiple whey acidic protein (WAP) domain containing proteins which are components of host immunity. In the present study, a new double WAP domain containing protein was identified from red swamp crayfish Procambarus clarkii, designated Pc-DWD. The ORF is 387 bp, encoding 128 ami...

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles

PubMed Central

Ray, Greeshma; Schmitt, Phuong Tieu

2016-01-01

ABSTRACT Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. IMPORTANCE Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles.

PubMed

Ray, Greeshma; Schmitt, Phuong Tieu; Schmitt, Anthony P

2016-01-20

Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable

The Armadillo Repeat Protein PF16 Is Essential for Flagellar Structure and Function in Plasmodium Male Gametes

PubMed Central

Ferguson, David J. P.; Bunting, Karen A.; Xu, Zhengyao; Bailes, Elizabeth; Sinden, Robert E.; Holder, Anthony A.; Smith, Elizabeth F.; Coates, Juliet C.; Rita Tewari

2010-01-01

Malaria, caused by the apicomplexan parasite Plasmodium, threatens 40% of the world's population. Transmission between vertebrate and insect hosts depends on the sexual stages of the life-cycle. The male gamete of Plasmodium parasite is the only developmental stage that possesses a flagellum. Very little is known about the identity or function of proteins in the parasite's flagellar biology. Here, we characterise a Plasmodium PF16 homologue using reverse genetics in the mouse malaria parasite Plasmodium berghei. PF16 is a conserved Armadillo-repeat protein that regulates flagellar structure and motility in organisms as diverse as green algae and mice. We show that P. berghei PF16 is expressed in the male gamete flagellum, where it plays a crucial role maintaining the correct microtubule structure in the central apparatus of the axoneme as studied by electron microscopy. Disruption of the PF16 gene results in abnormal flagellar movement and reduced fertility, but does not lead to complete sterility, unlike pf16 mutations in other organisms. Using homology modelling, bioinformatics analysis and complementation studies in Chlamydomonas, we show that some regions of the PF16 protein are highly conserved across all eukaryotes, whereas other regions may have species-specific functions. PF16 is the first ARM-repeat protein characterised in the malaria parasite genus Plasmodium and this study opens up a novel model for analysis of Plasmodium flagellar biology that may provide unique insights into an ancient organelle and suggest novel intervention strategies to control the malaria parasite. PMID:20886115

A new yeast gene with a myosin-like heptad repeat structure.

PubMed

Kölling, R; Nguyen, T; Chen, E Y; Botstein, D

1993-03-01

We isolated a gene encoding a 218 kDa myosin-like protein from Saccharomyces cerevisiae using a monoclonal antibody directed against human platelet myosin as a probe. The protein sequence encoded by the MLP1 gene (for myosin-like protein) contains extensive stretches of a heptad-repeat pattern suggesting that the protein can form coiled coils typical of myosins. Immunolocalization experiments using affinity-purified antibodies raised against a TrpE-MLP1 fusion protein showed a dot-like structure adjacent to the nucleus in yeast cells bearing the MLP1 gene on a multicopy plasmid. In mouse epithelial cells the yeast anti-MLP1 antibodies stained the nucleus. Mutants bearing disruptions of the MLP1 gene were viable, but more sensitive to ultraviolet light than wild-type strains, suggesting an involvement of MLP1 in DNA repair. The MLP1 gene was mapped to chromosome 11, 25 cM from met1.

Evolution of specifier proteins in glucosinolate-containing plants

PubMed Central

2012-01-01

Background The glucosinolate-myrosinase system is an activated chemical defense system found in plants of the Brassicales order. Glucosinolates are stored separately from their hydrolytic enzymes, the myrosinases, in plant tissues. Upon tissue damage, e.g. by herbivory, glucosinolates and myrosinases get mixed and glucosinolates are broken down to an array of biologically active compounds of which isothiocyanates are toxic to a wide range of organisms. Specifier proteins occur in some, but not all glucosinolate-containing plants and promote the formation of biologically active non-isothiocyanate products upon myrosinase-catalyzed glucosinolate breakdown. Results Based on a phytochemical screening among representatives of the Brassicales order, we selected candidate species for identification of specifier protein cDNAs. We identified ten specifier proteins from a range of species of the Brassicaceae and assigned each of them to one of the three specifier protein types (NSP, nitrile-specifier protein, ESP, epithiospecifier protein, TFP, thiocyanate-forming protein) after heterologous expression in Escherichia coli. Together with nine known specifier proteins and three putative specifier proteins found in databases, we subjected the newly identified specifier proteins to phylogenetic analyses. Specifier proteins formed three major clusters, named AtNSP5-cluster, AtNSP1-cluster, and ESP/TFP cluster. Within the ESP/TFP cluster, specifier proteins grouped according to the Brassicaceae lineage they were identified from. Non-synonymous vs. synonymous substitution rate ratios suggested purifying selection to act on specifier protein genes. Conclusions Among specifier proteins, NSPs represent the ancestral activity. The data support a monophyletic origin of ESPs from NSPs. The split between NSPs and ESPs/TFPs happened before the radiation of the core Brassicaceae. Future analyses have to show if TFP activity evolved from ESPs at least twice independently in different

Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins.

PubMed

Nick Pace, C; Huyghues-Despointes, Beatrice M P; Fu, Hailong; Takano, Kazufumi; Scholtz, J Martin; Grimsley, Gerald R

2010-05-01

The goal of this article is to gain a better understanding of the denatured state ensemble (DSE) of proteins through an experimental and computational study of their denaturation by urea. Proteins unfold to different extents in urea and the most hydrophobic proteins have the most compact DSE and contain almost as much secondary structure as folded proteins. Proteins that unfold to the greatest extent near pH 7 still contain substantial amounts of secondary structure. At low pH, the DSE expands due to charge-charge interactions and when the net charge per residue is high, most of the secondary structure is disrupted. The proteins in the DSE appear to contain substantial amounts of polyproline II conformation at high urea concentrations. In all cases considered, including staph nuclease, the extent of unfolding by urea can be accounted for using the data and approach developed in the laboratory of Wayne Bolen (Auton et al., Proc Natl Acad Sci 2007; 104:15317-15323).

Highly sensitive detection of individual HEAT and ARM repeats with HHpred and COACH.

PubMed

Kippert, Fred; Gerloff, Dietlind L

2009-09-24

HEAT and ARM repeats occur in a large number of eukaryotic proteins. As these repeats are often highly diverged, the prediction of HEAT or ARM domains can be challenging. Except for the most clear-cut cases, identification at the individual repeat level is indispensable, in particular for determining domain boundaries. However, methods using single sequence queries do not have the sensitivity required to deal with more divergent repeats and, when applied to proteins with known structures, in some cases failed to detect a single repeat. Testing algorithms which use multiple sequence alignments as queries, we found two of them, HHpred and COACH, to detect HEAT and ARM repeats with greatly enhanced sensitivity. Calibration against experimentally determined structures suggests the use of three score classes with increasing confidence in the prediction, and prediction thresholds for each method. When we applied a new protocol using both HHpred and COACH to these structures, it detected 82% of HEAT repeats and 90% of ARM repeats, with the minimum for a given protein of 57% for HEAT repeats and 60% for ARM repeats. Application to bona fide HEAT and ARM proteins or domains indicated that similar numbers can be expected for the full complement of HEAT/ARM proteins. A systematic screen of the Protein Data Bank for false positive hits revealed their number to be low, in particular for ARM repeats. Double false positive hits for a given protein were rare for HEAT and not at all observed for ARM repeats. In combination with fold prediction and consistency checking (multiple sequence alignments, secondary structure prediction, and position analysis), repeat prediction with the new HHpred/COACH protocol dramatically improves prediction in the twilight zone of fold prediction methods, as well as the delineation of HEAT/ARM domain boundaries. A protocol is presented for the identification of individual HEAT or ARM repeats which is straightforward to implement. It provides high

Highly Sensitive Detection of Individual HEAT and ARM Repeats with HHpred and COACH

PubMed Central

Kippert, Fred; Gerloff, Dietlind L.

2009-01-01

Background HEAT and ARM repeats occur in a large number of eukaryotic proteins. As these repeats are often highly diverged, the prediction of HEAT or ARM domains can be challenging. Except for the most clear-cut cases, identification at the individual repeat level is indispensable, in particular for determining domain boundaries. However, methods using single sequence queries do not have the sensitivity required to deal with more divergent repeats and, when applied to proteins with known structures, in some cases failed to detect a single repeat. Methodology and Principal Findings Testing algorithms which use multiple sequence alignments as queries, we found two of them, HHpred and COACH, to detect HEAT and ARM repeats with greatly enhanced sensitivity. Calibration against experimentally determined structures suggests the use of three score classes with increasing confidence in the prediction, and prediction thresholds for each method. When we applied a new protocol using both HHpred and COACH to these structures, it detected 82% of HEAT repeats and 90% of ARM repeats, with the minimum for a given protein of 57% for HEAT repeats and 60% for ARM repeats. Application to bona fide HEAT and ARM proteins or domains indicated that similar numbers can be expected for the full complement of HEAT/ARM proteins. A systematic screen of the Protein Data Bank for false positive hits revealed their number to be low, in particular for ARM repeats. Double false positive hits for a given protein were rare for HEAT and not at all observed for ARM repeats. In combination with fold prediction and consistency checking (multiple sequence alignments, secondary structure prediction, and position analysis), repeat prediction with the new HHpred/COACH protocol dramatically improves prediction in the twilight zone of fold prediction methods, as well as the delineation of HEAT/ARM domain boundaries. Significance A protocol is presented for the identification of individual HEAT or ARM repeats

Elfin: An algorithm for the computational design of custom three-dimensional structures from modular repeat protein building blocks.

PubMed

Yeh, Chun-Ting; Brunette, T J; Baker, David; McIntosh-Smith, Simon; Parmeggiani, Fabio

2018-02-01

Computational protein design methods have enabled the design of novel protein structures, but they are often still limited to small proteins and symmetric systems. To expand the size of designable proteins while controlling the overall structure, we developed Elfin, a genetic algorithm for the design of novel proteins with custom shapes using structural building blocks derived from experimentally verified repeat proteins. By combining building blocks with compatible interfaces, it is possible to rapidly build non-symmetric large structures (>1000 amino acids) that match three-dimensional geometric descriptions provided by the user. A run time of about 20min on a laptop computer for a 3000 amino acid structure makes Elfin accessible to users with limited computational resources. Protein structures with controlled geometry will allow the systematic study of the effect of spatial arrangement of enzymes and signaling molecules, and provide new scaffolds for functional nanomaterials. Copyright © 2017 Elsevier Inc. All rights reserved.

Roles of CUB and LDL receptor class A domain repeats of a transmembrane serine protease matriptase in its zymogen activation

PubMed Central

Inouye, Kuniyo; Tomoishi, Marie; Yasumoto, Makoto; Miyake, Yuka; Kojima, Kenji; Tsuzuki, Satoshi; Fushiki, Tohru

2013-01-01

Matriptase is a type II transmembrane serine protease containing two complement proteases C1r/C1s–urchin embryonic growth factor–bone morphogenetic protein domains (CUB repeat) and four low-density lipoprotein receptor class A domains (LDLRA repeat). The single-chain zymogen of matriptase has been found to exhibit substantial protease activity, possibly causing its own activation (i.e. conversion to a disulfide-linked two-chain fully active form), although the activation seems to be mediated predominantly by two-chain molecules. Our aim was to assess the roles of CUB and LDLRA repeats in zymogen activation. Transient expression studies of soluble truncated constructs of recombinant matriptase in COS-1 cells showed that the CUB repeat had an inhibitory effect on zymogen activation, possibly because it facilitated the interaction of two-chain molecules with a matriptase inhibitor, hepatocyte growth factor activator inhibitor type-1. By contrast, the LDLRA repeat had a promoting effect on zymogen activation. The effect of the LDLRA repeat seems to reflect its ability to increase zymogen activity. The proteolytic activities were higher in pseudozymogen forms of recombinant matriptase containing the LDLRA repeat than in a pseudozymogen without the repeat. Our findings provide new insights into the roles of these non-catalytic domains in the generation of active matriptase. PMID:23038671

Biophysical characterization of soluble Pseudomonas syringae ice nucleation protein InaZ fragments.

PubMed

Han, Yu Jin; Song, HyoJin; Lee, Chang Woo; Ly, Nguyễn Hoàng; Joo, Sang-Woo; Lee, Jun Hyuck; Kim, Soon-Jong; Park, SangYoun

2017-01-01

Ice nucleation protein (INP) with its functional domain consisting of multiple 48-residue repeat units effectively induces super-cooled water into ice. Circular dichroism and infrared deconvolution analyses on a soluble 240-residue fragment of Pseudomonas syringae InaZ (InaZ240) containing five 48-residue repeat units indicated that it is mostly composed of β-sheet and random coil. Analytical ultracentrifugation suggested that InaZ240 behaves as a monomer of an elongated ellipsoid. However, InaZ240 showed only minimum ice binding compared to anti-freeze proteins. Other P. syringae InaZ proteins with more 48-residue repeat units were made, in which the largest soluble fragment obtainable was an InaZ with twelve 48-residue repeat units. Size-exclusion chromatography analyses further suggested that the overall shape of the expressed InaZ fragments is pH-dependent, which becomes compact as the numbers of 48-residue repeat unit increase. Copyright © 2016 Elsevier B.V. All rights reserved.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2010-09-07

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2012-07-10

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Novel isoprenylated proteins identified by an expression library screen.

PubMed

Biermann, B J; Morehead, T A; Tate, S E; Price, J R; Randall, S K; Crowell, D N

1994-10-14

Isoprenylated proteins are involved in eukaryotic cell growth and signal transduction. The protein determinant for prenylation is a short carboxyl-terminal motif containing a cysteine, to which the isoprenoid is covalently attached via thioether linkage. To date, isoprenylated proteins have almost all been identified by demonstrating the attachment of an isoprenoid to previously known proteins. Thus, many isoprenylated proteins probably remain undiscovered. To identify novel isoprenylated proteins for subsequent biochemical study, colony blots of a Glycine max cDNA expression library were [3H]farnesyl-labeled in vitro. Proteins identified by this screen contained several different carboxyl termini that conform to consensus farnesylation motifs. These proteins included known farnesylated proteins (DnaJ homologs) and several novel proteins, two of which contained six or more tandem repeats of a hexapeptide having the consensus sequence (E/G)(G/P)EK(P/K)K. Thus, plants contain a diverse array of genes encoding farnesylated proteins, and our results indicate that fundamental differences in the identities of farnesylated proteins may exist between plants and other eukaryotes. Expression library screening by direct labeling can be adapted to identify isoprenylated proteins from other organisms, as well as proteins with other post-translational modifications.

Repeated pulses of serotonin required for long-term facilitation activate mitogen-activated protein kinase in sensory neurons of Aplysia

PubMed Central

Michael, Dan; Martin, Kelsey C.; Seger, Rony; Ning, Ming-Ming; Baston, Rene; Kandel, Eric R.

1998-01-01

Long-term facilitation of the connections between the sensory and motor neurons of the gill-withdrawal reflex in Aplysia requires five repeated pulses of serotonin (5-HT). The repeated pulses of 5-HT initiate a cascade of gene activation that leads ultimately to the growth of new synaptic connections. Several genes in this process have been identified, including the transcriptional regulators apCREB-1, apCREB-2, apC/EBP, and the cell adhesion molecule apCAM, which is thought to be involved in the formation of new synaptic connections. Here we report that the transcriptional regulators apCREB-2 and apC/EBP, as well as a peptide derived from the cytoplasmic domain of apCAM, are phosphorylated in vitro by Aplysia mitogen-activated protein kinase (apMAPK). We have cloned the cDNA encoding apMAPK and show that apMAPK activity is increased in sensory neurons treated with repeated pulses of 5-HT and by the cAMP pathway. These results suggest that apMAPK may participate with cAMP-dependent protein kinase during long-term facilitation in sensory cells by modifying some of the key elements involved in the consolidation of short- to long-lasting changes in synaptic strength. PMID:9465108

Leucine-Rich Repeat Kinase 2 Binds to Neuronal Vesicles through Protein Interactions Mediated by Its C-Terminal WD40 Domain

PubMed Central

Piccoli, Giovanni; Onofri, Franco; Cirnaru, Maria Daniela; Kaiser, Christoph J. O.; Jagtap, Pravinkumar; Kastenmüller, Andreas; Pischedda, Francesca; Marte, Antonella; von Zweydorf, Felix; Vogt, Andreas; Giesert, Florian; Pan, Lifeng; Antonucci, Flavia; Kiel, Christina; Zhang, Mingjie; Weinkauf, Sevil; Sattler, Michael; Sala, Carlo; Matteoli, Michela; Ueffing, Marius

2014-01-01

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function. PMID:24687852

[Bioinformatics Analysis of Clustered Regularly Interspaced Short Palindromic Repeats in the Genomes of Shigella].

PubMed

Wang, Pengfei; Wang, Yingfang; Duan, Guangcai; Xue, Zerun; Wang, Linlin; Guo, Xiangjiao; Yang, Haiyan; Xi, Yuanlin

2015-04-01

This study was aimed to explore the features of clustered regularly interspaced short palindromic repeats (CRISPR) structures in Shigella by using bioinformatics. We used bioinformatics methods, including BLAST, alignment and RNA structure prediction, to analyze the CRISPR structures of Shigella genomes. The results showed that the CRISPRs existed in the four groups of Shigella, and the flanking sequences of upstream CRISPRs could be classified into the same group with those of the downstream. We also found some relatively conserved palindromic motifs in the leader sequences. Repeat sequences had the same group with corresponding flanking sequences, and could be classified into two different types by their RNA secondary structures, which contain "stem" and "ring". Some spacers were found to homologize with part sequences of plasmids or phages. The study indicated that there were correlations between repeat sequences and flanking sequences, and the repeats might act as a kind of recognition mechanism to mediate the interaction between foreign genetic elements and Cas proteins.

Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

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

Peng, Jamy C.

Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) thatmore » binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those

Identification of the chemical constituents of Chinese medicine Yi-Xin-Shu capsule by molecular feature orientated precursor ion selection and tandem mass spectrometry structure elucidation.

PubMed

Wang, Hong-ping; Chen, Chang; Liu, Yan; Yang, Hong-Jun; Wu, Hong-Wei; Xiao, Hong-Bin

2015-11-01

The incomplete identification of the chemical components of traditional Chinese medicinal formula has been one of the bottlenecks in the modernization of traditional Chinese medicine. Tandem mass spectrometry has been widely used for the identification of chemical substances. Current automatic tandem mass spectrometry acquisition, where precursor ions were selected according to their signal intensity, encounters a drawback in chemical substances identification when samples contain many overlapping signals. Compounds in minor or trace amounts could not be identified because most tandem mass spectrometry information was lost. Herein, a molecular feature orientated precursor ion selection and tandem mass spectrometry structure elucidation method for complex Chinese medicine chemical constituent analysis was developed. The precursor ions were selected according to their two-dimensional characteristics of retention times and mass-to-charge ratio ranges from herbal compounds, so that all precursor ions from herbal compounds were included and more minor chemical constituents in Chinese medicine were identified. Compared to the conventional automatic tandem mass spectrometry setups, the approach is novel and can overcome the drawback for chemical substances identification. As an example, 276 compounds from the Chinese Medicine of Yi-Xin-Shu capsule were identified. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ermelin, an endoplasmic reticulum transmembrane protein, contains the novel HELP domain conserved in eukaryotes.

PubMed

Suzuki, Akiko; Endo, Takeshi

2002-02-06

We have cloned a cDNA encoding a novel protein referred to as ermelin from mouse C2 skeletal muscle cells. This protein contained six hydrophobic amino acid stretches corresponding to transmembrane domains, two histidine-rich sequences, and a sequence homologous to the fusion peptides of certain fusion proteins. Ermelin also contained a novel modular sequence, designated as HELP domain, which was highly conserved among eukaryotes, from yeast to higher plants and animals. All these HELP domain-containing proteins, including mouse KE4, Drosophila Catsup, and Arabidopsis IAR1, possessed multipass transmembrane domains and histidine-rich sequences. Ermelin was predominantly expressed in brain and testis, and induced during neuronal differentiation of N1E-115 neuroblastoma cells but downregulated during myogenic differentiation of C2 cells. The mRNA was accumulated in hippocampus and cerebellum of brain and central areas of seminiferous tubules in testis. Epitope-tagging experiments located ermelin and KE4 to a network structure throughout the cytoplasm. Staining with the fluorescent dye DiOC(6)(3) identified this structure as the endoplasmic reticulum. These results suggest that at least some, if not all, of the HELP domain-containing proteins are multipass endoplasmic reticulum membrane proteins with functions conserved among eukaryotes.

Quality evaluation of Guan-Xin-Ning injection based on fingerprint analysis and simultaneous separation and determination of seven bioactive constituents by capillary electrophoresis.

PubMed

Xu, Liying; Chang, Ruimiao; Chen, Meng; Li, Lou; Huang, Yayun; Zhang, Hongfen; Chen, Anjia

2017-12-01

The purpose of this study was to develop a comprehensive, rapid and practical capillary electrophoresis (CE) method for quality control (QC) of Guan-Xin-Ning (GXN) injection based on fingerprint analysis and simultaneous separation and determination of seven constituents. In fingerprint analysis, a capillary zone electrophoresis (CZE) method with a running buffer of 30 mM borate solution (pH 9.3) was established. Meanwhile, ten batches of samples were used to establish the fingerprint electropherogram and 34 common peaks were obtained within 20 min. The RSD of relative migration times (RMT) and relative peak areas (RPA) were less than 5%. In order to further evaluate the quality of GXN injection, a micellar electrokinetic chromatography (MEKC) method was developed for simultaneous separation and determination of bioactive constituents. Seven components reached baseline separation with a running buffer containing 35 mM SDS and 45 mM borate solution (pH 9.3). A good linearity was obtained with correlation coefficients from 0.9906 to 0.9997. The LOD and LOQ ranged from 0.12 to 1.50 μg/mL and from 0.40 to 4.90 μg/mL, respectively. The recoveries ranged between 99.0 and 104.4%. Therefore, it was concluded that the proposed method can be used for full-scale quality analysis of GXN injection. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Histone Database: an integrated resource for histones and histone fold-containing proteins

PubMed Central

Mariño-Ramírez, Leonardo; Levine, Kevin M.; Morales, Mario; Zhang, Suiyuan; Moreland, R. Travis; Baxevanis, Andreas D.; Landsman, David

2011-01-01

Eukaryotic chromatin is composed of DNA and protein components—core histones—that act to compactly pack the DNA into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleosomes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifications and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones (H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current information on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of chromatin structure and function focused on histones and histone fold-containing proteins. Database URL: The Histone Sequence Database is freely available and can be accessed at http://research.nhgri.nih.gov/histones/. PMID:22025671

Protein profile of Lupinus texensis phloem sap exudates: searching for Fe- and Zn-containing proteins.

PubMed

Lattanzio, Giuseppe; Andaluz, Sofía; Matros, Andrea; Calvete, Juan José; Kehr, Julia; Abadía, Anunciación; Abadía, Javier; López-Millán, Ana-Flor

2013-08-01

The aim of this study was to obtain a comprehensive overview of the phloem sap protein profile of Lupinus texensis, with a special focus on proteins binding Fe and Zn. L. texensis was chosen as model plant given the simplicity to obtain exudates from sieve elements. Protein profiling by 2DE revealed 249 spots, and 54 of them were unambiguously identified by MALDI-MS and ESI-MS/MS. The largest number of identified protein species belongs to protein modification/turnover and general metabolism (19-21%), followed by redox homeostasis (9%) and defense and cell structural components (7%). This protein profile is similar to that reported in other plant species, suggesting that the phloem sap proteome is quite conserved. Staining of 2DE gels for Fe-containing proteins and affinity chromatography experiments revealed the presence of two low molecular weight Fe-binding proteins in phloem sap: a metallothionein-like protein type 2B identified in the Fe-affinity chromatography, and a second protein identified with both Fe staining methods. This protein species had a molecular weight of 13.5 kDa, a pI of 5.6 and 51% homology to a phloem-specific protein from Medicago truncatula. Zinc affinity chromatography revealed four Zn-binding proteins in phloem sap, one belonging to the dehydrin family and three Zn finger proteins. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates

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

Jackson, P.J.; Walthers, E.A.; Richmond, K.L.

1997-04-01

PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five Polymorphisms differed by the presence Of two to six copies of the 12-bp tandem repeat 5{prime}-CAATATCAACAA-3{prime}. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats aremore » generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations. 22 refs., 4 figs., 3 tabs.« less

Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca[superscript 2+]-dependent Double-stranded DNA Binding Activity

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

Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 {angstrom} tetrameric ring structure. The inner circle ofmore » the Csn2 tetrameric ring is {approx}26 {angstrom} wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an {alpha}/{beta} domain and an {alpha}-helical domain; significant hinge motion was observed between these two domains. Ca{sup 2+} was located at strategic positions in the oligomerization interface. We further showed that removal of Ca{sup 2+} ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca{sup 2+} ions.« less

Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography—Tandem Mass Spectrometry

PubMed Central

Tabb, David L.; Vega-Montoto, Lorenzo; Rudnick, Paul A.; Variyath, Asokan Mulayath; Ham, Amy-Joan L.; Bunk, David M.; Kilpatrick, Lisa E.; Billheimer, Dean D.; Blackman, Ronald K.; Cardasis, Helene L.; Carr, Steven A.; Clauser, Karl R.; Jaffe, Jacob D.; Kowalski, Kevin A.; Neubert, Thomas A.; Regnier, Fred E.; Schilling, Birgit; Tegeler, Tony J.; Wang, Mu; Wang, Pei; Whiteaker, Jeffrey R.; Zimmerman, Lisa J.; Fisher, Susan J.; Gibson, Bradford W.; Kinsinger, Christopher R.; Mesri, Mehdi; Rodriguez, Henry; Stein, Steven E.; Tempst, Paul; Paulovich, Amanda G.; Liebler, Daniel C.; Spiegelman, Cliff

2009-01-01

The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35–60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2013-03-12

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetase that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel sythetases molecules, methods for identifying and making the novel synthetases, methods for producing containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lapidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes.

PubMed

Al-Attar, Sinan; Westra, Edze R; van der Oost, John; Brouns, Stan J J

2011-04-01

Many prokaryotes contain the recently discovered defense system against mobile genetic elements. This defense system contains a unique type of repetitive DNA stretches, termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs). CRISPRs consist of identical repeated DNA sequences (repeats), interspaced by highly variable sequences referred to as spacers. The spacers originate from either phages or plasmids and comprise the prokaryotes' 'immunological memory'. CRISPR-associated (cas) genes encode conserved proteins that together with CRISPRs make-up the CRISPR/Cas system, responsible for defending the prokaryotic cell against invaders. CRISPR-mediated resistance has been proposed to involve three stages: (i) CRISPR-Adaptation, the invader DNA is encountered by the CRISPR/Cas machinery and an invader-derived short DNA fragment is incorporated in the CRISPR array. (ii) CRISPR-Expression, the CRISPR array is transcribed and the transcript is processed by Cas proteins. (iii) CRISPR-Interference, the invaders' nucleic acid is recognized by complementarity to the crRNA and neutralized. An application of the CRISPR/Cas system is the immunization of industry-relevant prokaryotes (or eukaryotes) against mobile-genetic invasion. In addition, the high variability of the CRISPR spacer content can be exploited for phylogenetic and evolutionary studies. Despite impressive progress during the last couple of years, the elucidation of several fundamental details will be a major challenge in future research.

Myricetin Reduces Toxic Level of CAG Repeats RNA in Huntington's Disease (HD) and Spino Cerebellar Ataxia (SCAs).

PubMed

Khan, Eshan; Tawani, Arpita; Mishra, Subodh Kumar; Verma, Arun Kumar; Upadhyay, Arun; Kumar, Mohit; Sandhir, Rajat; Mishra, Amit; Kumar, Amit

2018-01-19

Huntington's disease (HD) is a neurodegenerative disorder that is caused by abnormal expansion of CAG repeats in the HTT gene. The transcribed mutant RNA contains expanded CAG repeats that translate into a mutant huntingtin protein. This expanded CAG repeat also causes mis-splicing of pre-mRNA due to sequestration of muscle blind like-1 splicing factor (MBNL1), and thus both of these elicit the pathogenesis of HD. Targeting the onset as well as progression of HD by small molecules could be a potent therapeutic approach. We have screened a set of small molecules to target this transcript and found Myricetin, a flavonoid, as a lead molecule that interacts with the CAG motif and thus prevents the translation of mutant huntingtin protein as well as sequestration of MBNL1. Here, we report the first solution structure of the complex formed between Myricetin and RNA containing the 5'CAG/3'GAC motif. Myricetin interacts with this RNA via base stacking at the AA mismatch. Moreover, Myricetin was also found reducing the proteo-toxicity generated due to the aggregation of polyglutamine, and further, its supplementation also improves neurobehavioral deficits in the HD mouse model. Our study provides the structural and mechanistic basis of Myricetin as an effective therapeutic candidate for HD and other polyQ related disorders.

The transport of Staufen2-containing ribonucleoprotein complexes involves kinesin motor protein and is modulated by mitogen-activated protein kinase pathway.

PubMed

Jeong, Ji-Hye; Nam, Yeon-Ju; Kim, Seok-Yong; Kim, Eung-Gook; Jeong, Jooyoung; Kim, Hyong Kyu

2007-09-01

There is increasing evidence showing that mRNA is transported to the neuronal dendrites in ribonucleoprotein (RNP) complexes or RNA granules, which are aggregates of mRNA, rRNA, ribosomal proteins, and RNA-binding proteins. In these RNP complexes, Staufen, a double-stranded RNA-binding protein, is believed to be a core component that plays a key role in the dendritic mRNA transport. This study investigated the molecular mechanisms of the dendritic mRNA transport using green fluorescent protein-tagged Staufen2 produced employing a Sindbis viral expression system. The kinesin heavy chain was found to be associated with Staufen2. The inhibition of kinesin resulted in a significant decrease in the level of dendritic transport of the Staufen2-containing RNP complexes in neurons under non-stimulating or stimulating conditions. This suggests that the dendritic transport of the Staufen2-containing RNP complexes use kinesin as a motor protein. A mitogen-activated protein kinase inhibitor, PD98059, inhibited the activity-induced increase in the amount of both the Staufen2-containing RNP complexes and Ca(2+)/calmodulin-dependent protein kinase II alpha-subunit mRNA in the distal dendrites of cultured hippocampal neurons. Overall, these results suggest that dendritic mRNA transport is mediated via the Staufen2 and kinesin motor proteins and might be modulated by the neuronal activity and mitogen-activated protein kinase pathway.

New valve and bonding designs for microfluidic biochips containing proteins.

PubMed

Lu, Chunmeng; Xie, Yubing; Yang, Yong; Cheng, Mark M-C; Koh, Chee-Guan; Bai, Yunling; Lee, L James; Juang, Yi-Je

2007-02-01

Two major concerns in the design and fabrication of microfluidic biochips are protein binding on the channel surface and protein denaturing during device assembly. In this paper, we describe new methods to solve these problems. A "fishbone" microvalve design based on the concept of superhydrophobicity was developed to replace the capillary valve in applications where the chip surface requires protein blocking to prevent nonspecific binding. Our experimental results show that the valve functions well in a CD-like ELISA device. The packaging of biochips containing pre-loaded proteins is also a challenging task since conventional sealing methods often require the use of high temperatures, electric voltages, or organic solvents that are detrimental to the protein activity. Using CO2 gas to enhance the diffusion of polymer molecules near the device surface can result in good bonding at low temperatures and low pressure. This bonding method has little influence on the activity of the pre-loaded proteins after bonding.

UBXD4, a UBX-containing protein, regulates the cell surface number and stability of alpha3-containing nicotinic acetylcholine receptors.

PubMed

Rezvani, Khosrow; Teng, Yanfen; Pan, Yaping; Dani, John A; Lindstrom, Jon; García Gras, Eduardo A; McIntosh, J Michael; De Biasi, Mariella

2009-05-27

Adaptor proteins are likely to modulate spatially and temporally the trafficking of a number of membrane proteins, including neuronal nicotinic acetylcholine receptors (nAChRs). A yeast two-hybrid screen identified a novel UBX-containing protein, UBXD4, as one of the cytosolic proteins that interact directly with the alpha3 and alpha4 nAChR subunits. The function of UBX-containing proteins is largely unknown. Immunoprecipitation and confocal microscopy confirmed the interaction of UBXD4 with alpha3-containing nAChRs (alpha3* nAChRs) expressed in HEK293 cells, PC12 cells, and rat cortical neurons. Overexpression of UBXD4 in differentiated PC12 cells (dPC12) increased nAChR cell surface expression, especially that of the alpha3beta2 subtype. These findings were corroborated by electrophysiology, immunofluorescent staining, and biotinylation of surface receptors. Silencing of UBXD4 led to a significant reduction of alpha3* nAChRs in rat cortical neurons and dPC12 cells. Biochemical and immunofluorescence studies of endogenous UBXD4 showed that the protein is located in both the ER and cis-Golgi compartments. Our investigations also showed that the alpha3 subunit is ubiquitinated and that UBXD4 can interfere with its ubiquitination and consequent degradation by the proteasome. Our data suggest that UBXD4 modulates the distribution of alpha3* nAChRs between specialized intracellular compartments and the plasma membrane. This effect is achieved by controlling the stability of the alpha3 subunit and, consequently, the number of receptors at the cell surface.

Two synthetic Sp1-binding sites functionally substitute for the 21-base-pair repeat region to activate simian virus 40 growth in CV-1 cells.

PubMed Central

Lednicky, J; Folk, W R

1992-01-01

The 21-bp repeat region of simian virus 40 (SV40) activates viral transcription and DNA replication and contains binding sites for many cellular proteins, including Sp1, LSF, ETF, Ap2, Ap4, GT-1B, H16, and p53, and for the SV40 large tumor antigen. We have attempted to reduce the complexity of this region while maintaining its growth-promoting capacity. Deletion of the 21-bp repeat region from the SV40 genome delays the expression of viral early proteins and DNA replication and reduces virus production in CV-1 cells. Replacement of the 21-bp repeat region with two copies of DNA sequence motifs bound with high affinities by Sp1 promotes SV40 growth in CV-1 cells to nearly wild-type levels, but substitution by motifs bound less avidly by Sp1 or bound by other activator proteins does not restore growth. This indicates that Sp1 or a protein with similar sequence specificity is primarily responsible for the function of the 21-bp repeat region. We speculate about how Sp1 activates both SV40 transcription and DNA replication. Images PMID:1328672

m-Trifluoromethyl-diphenyl Diselenide Regulates Prefrontal Cortical MOR and KOR Protein Levels and Abolishes the Phenotype Induced by Repeated Forced Swim Stress in Mice.

PubMed

Rosa, Suzan Gonçalves; Pesarico, Ana Paula; Martini, Franciele; Nogueira, Cristina Wayne

2018-04-05

The present study aimed to investigate the m-trifluoromethyl-diphenyl diselenide [(m-CF 3 -PhSe) 2 ] effects on prefrontal cortical MOR and KOR protein levels and phenotype induced by repeated forced swim stress (FSS) in mice. Adult Swiss mice were subjected to repeated FSS sessions, and after that, they performed the spontaneous locomotor/exploratory activity, tail suspension, and splash tests. (m-CF 3 -PhSe) 2 (0.1 to 5 mg/kg) was administered to mice 30 min before the first FSS session and 30 min before the subsequent repeated FSS. (m-CF 3 -PhSe) 2 abolished the phenotype induced by repeated FSS in mice. In addition, a single FSS session increased μ but reduced δ-opioid receptor contents, without changing the κ content. Mice subjected to repeated FSS had an increase in the μ content when compared to those of naïve group or subjected to single FSS. Repeated FSS induced an increase of δ-opioid receptor content compared to those mice subjected to single FSS. However, the δ-opioid receptor contents were lower than those found in the naïve group. The mice subjected to repeated FSS showed an increase in the κ-opioid receptor content when compared to that of the naïve mice. (m-CF 3 -PhSe) 2 regulated the protein contents of μ and κ receptors in mice subjected to repeated FSS. These findings demonstrate that (m-CF 3 -PhSe) 2 was effective to abolish the phenotype induced by FSS, which was accompanied by changes in the contents of cortical μ- and κ-opioid receptors.

Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein-Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis.

PubMed

Barreales, Eva G; Vicente, Cláudia M; de Pedro, Antonio; Santos-Aberturas, Javier; Aparicio, Jesús F

2018-05-15

The biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A Streptomyces antibiotic regulatory protein-large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene, pimM A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide. Here, we used promoter engineering and quantitative transcriptional analyses to determine the contributions of the different heptameric repeats to transcriptional activation and final polyene production. Optimized promoters have thus been developed. Deletion studies and electrophoretic mobility assays were used for the definition of DNA-binding boxes formed by 22-nucleotide sequences comprising two conserved heptameric direct repeats separated by four-nucleotide less conserved spacers. The cooperative binding of PimR SARP appears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient binding. IMPORTANCE Here, we have shown that a modulation of the production of the antifungal pimaricin in Streptomyces natalensis can be accomplished via promoter engineering of the PAS-LuxR transcriptional activator pimM The expression of this gene is

Cloning and identification of a cDNA that encodes a novel human protein with thrombospondin type I repeat domain, hPWTSR.

PubMed

Chen, Jin-Zhong; Wang, Shu; Tang, Rong; Yang, Quan-Sheng; Zhao, Enpeng; Chao, Yaoqiong; Ying, Kang; Xie, Yi; Mao, Yu-Min

2002-09-01

A cDNA was isolated from the fetal brain cDNA library by high throughput cDNA sequencing. The 2390 bp cDNA with an open reading fragment (ORF) of 816 bp encodes a 272 amino acids putative protein with a thrombospondin type I repeat (TSR) domain and a cysteine-rich region at the N-terminus, so it is named hPWTSR. We used Northern blot detected two bands with length of about 3 kb and 4 kb respectively, which expressed in human adult tissues with different intensities. The expression pattern was verified by RT-PCR, revealing that the transcripts were expressed ubiquitously in fetal tissues and human tumor tissues too. However, the transcript was detected neither in ovarian carcinoma GI-102 nor in lung carcinoma LX-1. Blast analysis against NCBI database revealed that the new gene contained at least 5 exons and located in human chromosome 6q22.33. Our results demonstrate that the gene is a novel member of TSR supergene family.

A diverse host thrombospondin-type-1 repeat protein repertoire promotes symbiont colonization during establishment of cnidarian-dinoflagellate symbiosis.

PubMed

Neubauer, Emilie-Fleur; Poole, Angela Z; Neubauer, Philipp; Detournay, Olivier; Tan, Kenneth; Davy, Simon K; Weis, Virginia M

2017-05-08

The mutualistic endosymbiosis between cnidarians and dinoflagellates is mediated by complex inter-partner signaling events, where the host cnidarian innate immune system plays a crucial role in recognition and regulation of symbionts. To date, little is known about the diversity of thrombospondin-type-1 repeat (TSR) domain proteins in basal metazoans or their potential role in regulation of cnidarian-dinoflagellate mutualisms. We reveal a large and diverse repertoire of TSR proteins in seven anthozoan species, and show that in the model sea anemone Aiptasia pallida the TSR domain promotes colonization of the host by the symbiotic dinoflagellate Symbiodinium minutum . Blocking TSR domains led to decreased colonization success, while adding exogenous TSRs resulted in a 'super colonization'. Furthermore, gene expression of TSR proteins was highest at early time-points during symbiosis establishment. Our work characterizes the diversity of cnidarian TSR proteins and provides evidence that these proteins play an important role in the establishment of cnidarian-dinoflagellate symbiosis.

Antidepressant effects of Kai-Xin-San in fluoxetine-resistant depression rats.

PubMed

Dong, X Z; Wang, D X; Lu, Y P; Yuan, S; Liu, P; Hu, Y

2017-08-17

This study aimed to investigate the antidepressant effect and the mechanism of action of Kai-Xin-San (KXS) in fluoxetine-resistant depressive (FRD) rats. Two hundred male Wistar rats weighing 200±10 g were exposed to chronic and unpredictable mild stresses (CUMS) for 4 weeks and given fluoxetine treatment simultaneously. The rats that did not show significant improvement in behavioral indexes were chosen as the FRD model rats. These rats were randomly divided into four groups: FRD model control; oral fluoxetine and aspirin; oral KXS at a dose of 338 mg·kg-1·day-1; and oral KXS at a dose of 676 mg·kg-1·day-1. Rats continued to be exposed to CUMS and underwent treatment once a day for 3 weeks, then cytokine (COX-2, IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, TGF-β, and TNF-α) levels in the hippocampus and serum, and organ coefficients were measured. Both doses of KXS improved the crossing and rearing frequencies, sucrose-preference index, and body weight in FRD rats. KXS at a dose of 338 mg·kg-1·day-1reduced COX-2, IL-2, IL-6, TNF-α levels, increased IL-10 level in the hippocampus, and reduced IL-2 and TNF-α levels in serum. KXS at a dose of 676 mg·kg-1·day-1reduced TNF-α level in the hippocampus, reduced IL-2 and TNF-α levels in serum, and increased IFN-γ and IL-10 levels in the hippocampus and serum. There were no significant differences in organ-coefficients of the spleen among and between groups. The results suggested that oral administration of KXS in FRD rats was effective in improving behavior disorders by influencing various inflammatory pathways.

Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry.

PubMed

Reiz, Bela; Li, Liang

2010-09-01

Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein. 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

Protein retention and liver aminotransferase activities in Atlantic salmon fed diets containing different energy sources

USGS Publications Warehouse

Fynn-Aikins, K.; Hughes, S.G.; Vandenberg, G.W.

1995-01-01

Atlantic salmon (Salmo salar) fingerlings (14.4 g) were fed diets containing either glucose, dextrin, raw corn starch and lipid, or a high protein U.S. Fish and Wildlife Service open-formula diet (ASD2-30) for 12 weeks. Significant differences in weight gain and feed: gain ratio were not observed among salmon fed the diets containing glucose, dextrin or ASD2-30. Diets containing dextrin and glucose supported greater protein retention and reduction in alanine aminotransferase activity than the other diets. Activity of aspartate aminotransferase was not affected by the dietary treatment. Protein retention correlated highly with alanine aminotransferase activity.

Protein design to understand peptide ligand recognition by tetratricopeptide repeat proteins.

PubMed

Cortajarena, Aitziber L; Kajander, Tommi; Pan, Weilan; Cocco, Melanie J; Regan, Lynne

2004-04-01

Protein design aims to understand the fundamentals of protein structure by creating novel proteins with pre-specified folds. An equally important goal is to understand protein function by creating novel proteins with pre-specified activities. Here we describe the design and characterization of a tetratricopeptide (TPR) protein, which binds to the C-terminal peptide of the eukaryotic chaperone Hsp90. The design emphasizes the importance of both direct, short-range protein-peptide interactions and of long-range electrostatic optimization. We demonstrate that the designed protein binds specifically to the desired peptide and discriminates between it and the similar C-terminal peptide of Hsp70.

CMsearch: simultaneous exploration of protein sequence space and structure space improves not only protein homology detection but also protein structure prediction.

PubMed

Cui, Xuefeng; Lu, Zhiwu; Wang, Sheng; Jing-Yan Wang, Jim; Gao, Xin

2016-06-15

Protein homology detection, a fundamental problem in computational biology, is an indispensable step toward predicting protein structures and understanding protein functions. Despite the advances in recent decades on sequence alignment, threading and alignment-free methods, protein homology detection remains a challenging open problem. Recently, network methods that try to find transitive paths in the protein structure space demonstrate the importance of incorporating network information of the structure space. Yet, current methods merge the sequence space and the structure space into a single space, and thus introduce inconsistency in combining different sources of information. We present a novel network-based protein homology detection method, CMsearch, based on cross-modal learning. Instead of exploring a single network built from the mixture of sequence and structure space information, CMsearch builds two separate networks to represent the sequence space and the structure space. It then learns sequence-structure correlation by simultaneously taking sequence information, structure information, sequence space information and structure space information into consideration. We tested CMsearch on two challenging tasks, protein homology detection and protein structure prediction, by querying all 8332 PDB40 proteins. Our results demonstrate that CMsearch is insensitive to the similarity metrics used to define the sequence and the structure spaces. By using HMM-HMM alignment as the sequence similarity metric, CMsearch clearly outperforms state-of-the-art homology detection methods and the CASP-winning template-based protein structure prediction methods. Our program is freely available for download from http://sfb.kaust.edu.sa/Pages/Software.aspx : xin.gao@kaust.edu.sa Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

Experiences from occupational exposure limits set on aerosols containing allergenic proteins.

PubMed

Nielsen, Gunnar D; Larsen, Søren T; Hansen, Jitka S; Poulsen, Lars K

2012-10-01

Occupational exposure limits (OELs) together with determined airborne exposures are used in risk assessment based managements of occupational exposures to prevent occupational diseases. In most countries, OELs have only been set for few protein-containing aerosols causing IgE-mediated allergies. They comprise aerosols of flour dust, grain dust, wood dust, natural rubber latex, and the subtilisins, which are proteolytic enzymes. These aerosols show dose-dependent effects and levels have been established, where nearly all workers may be exposed without adverse health effects, which are required for setting OELs. Our aim is to analyse prerequisites for setting OELs for the allergenic protein-containing aerosols. Opposite to the key effect of toxicological reactions, two thresholds, one for the sensitization phase and one for elicitation of IgE-mediated symptoms in sensitized individuals, are used in the OEL settings. For example, this was the case for flour dust, where OELs were based on dust levels due to linearity between flour dust and its allergen levels. The critical effects for flour and grain dust OELs were different, which indicates that conclusion by analogy (read-across) must be scientifically well founded. Except for subtilisins, no OEL have been set for other industrial enzymes, where many of which are high volume chemicals. For several of these, OELs have been proposed in the scientific literature during the last two decades. It is apparent that the scientific methodology is available for setting OELs for proteins and protein-containing aerosols where the critical effect is IgE sensitization and IgE-mediated airway diseases.

Experiences from Occupational Exposure Limits Set on Aerosols Containing Allergenic Proteins

PubMed Central

Nielsen, Gunnar D.

2012-01-01

Occupational exposure limits (OELs) together with determined airborne exposures are used in risk assessment based managements of occupational exposures to prevent occupational diseases. In most countries, OELs have only been set for few protein-containing aerosols causing IgE-mediated allergies. They comprise aerosols of flour dust, grain dust, wood dust, natural rubber latex, and the subtilisins, which are proteolytic enzymes. These aerosols show dose-dependent effects and levels have been established, where nearly all workers may be exposed without adverse health effects, which are required for setting OELs. Our aim is to analyse prerequisites for setting OELs for the allergenic protein-containing aerosols. Opposite to the key effect of toxicological reactions, two thresholds, one for the sensitization phase and one for elicitation of IgE-mediated symptoms in sensitized individuals, are used in the OEL settings. For example, this was the case for flour dust, where OELs were based on dust levels due to linearity between flour dust and its allergen levels. The critical effects for flour and grain dust OELs were different, which indicates that conclusion by analogy (read-across) must be scientifically well founded. Except for subtilisins, no OEL have been set for other industrial enzymes, where many of which are high volume chemicals. For several of these, OELs have been proposed in the scientific literature during the last two decades. It is apparent that the scientific methodology is available for setting OELs for proteins and protein-containing aerosols where the critical effect is IgE sensitization and IgE-mediated airway diseases. PMID:22843406

Chinese patent medicine Xin-Ke-Shu inhibits Ca2+ overload and dysfunction of fatty acid β-oxidation in rats with myocardial infarction induced by LAD ligation.

PubMed

Yang, Yong; Jia, Hongmei; Yu, Meng; Zhou, Chao; Sun, Lili; Zhao, Yang; Zhang, Hongwu; Zou, Zhongmei

2018-03-15

Myocardial infarction (MI) occurs during a sustained insufficient blood supply to the heart, eventually leading to myocardial necrosis. Xin-Ke-Shu tablet (XKS) is a prescription herbal compound and a patented medicine extensively used in the clinical treatment of coronary heart disease (CHD). To understand the molecular mechanism of the XKS action against MI in detail, it is necessary to investigate the altered metabolome and related pathways coincident with clinical features. In this study, tissue-targeted metabonomics based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) were developed to explore the metabolic changes associated with XKS treatment in the heart tissue of rats with MI induced by a left anterior descending coronary artery ligation (LAD). The metabolic disorder induced by LAD was alleviated after low-dose XKS (LD) and intermediate-dose XKS (MD) treatment. XKS modulated six perturbed metabolic pathways. Among them, inhibition of Ca 2+ overload and dysfunction of fatty acid β-oxidation-related metabolic pathways likely underlie the therapeutic effects of XKS against MI. In agreement with its observed effect on metabolite perturbation, XKS reversed the over-expression of the four key proteins, long-chain acyl-CoA synthetase 1 (ACSL1), carnitine palmitoyl transferase-1 (CPT1B), calcium/calmodulin-dependent kinase II (CaMKII), and phospholipase A2IIA (PLA2IIA). Both metabolite and protein changes suggested that XKS exerts its therapeutic effect on metabolic perturbations in LAD-induced MI mainly by inhibiting the Ca 2+ overload and fatty acid β-oxidation dysfunction. Copyright © 2018 Elsevier B.V. All rights reserved.

Large Polyglutamine Repeats Cause Muscle Degeneration in SCA17 Mice

PubMed Central

Huang, Shanshan; Yang, Su; Guo, Jifeng; Yan, Sen; Gaertig, Marta A.; Li, Shihua; Li, Xiao-Jiang

2015-01-01

SUMMARY In polyglutamine (polyQ) diseases, large polyQ repeats cause juvenile cases with different symptoms than adult-onset patients, who carry smaller expanded polyQ repeats. The mechanisms behind the differential pathology mediated by different polyQ repeat lengths remain unknown. By studying knock-in mouse models of spinal cerebellar ataxia-17 (SCA17), we found that a large polyQ (105 glutamines) in the TATA box-binding protein (TBP) preferentially causes muscle degeneration and reduces the expression of muscle-specific genes. Direct expression of TBP with different polyQ repeats in mouse muscle revealed that muscle degeneration is mediated only by the large polyQ repeats. Different polyQ repeats differentially alter TBP’s interaction with neuronal and muscle-specific transcription factors. As a result, the large polyQ repeat decreases the association of MyoD with TBP and DNA promoters. Our findings suggest that specific alterations in protein interactions by large polyQ repeats may account for the unique pathology in juvenile polyQ diseases. PMID:26387956

Biotin Attachment Domain-Containing Proteins Irreversibly Inhibit Acetyl CoA Carboxylase

DOE PAGES

Keereetaweep, Jantana; Liu, Hui; Zhai, Zhiyang; ...

2018-04-06

The first committed step in fatty acid synthesis is mediated by Acetyl-CoA carboxylase (ACCase), a biotin-dependent enzyme that carboxylates acetyl-CoA to produce malonyl-CoA. ACCase can be feedback-regulated by short-term (reversible) and longer-term (irreversible) inhibition upon oversupply of fatty acids (FA) provided by Tween80 (predominantly containing oleic acid; 18:1). Biotin-Attachment-Domain-Containing (BADC) proteins are inactive analogs of biotin carboxyl transfer protein (BCCP) that lack biotin and their incorporation into ACCase downregulates it by displacing active (biotin-containing) BCCP subunits. Individual T-DNA insertion lines of BADC1, BADC2, and BADC3 were used to generate badc1badc2 and badc1badc3. The badc1badc3 mutant and wild-type exhibited normal growthmore » and development, however ACCase activity was 26% higher in badc1badc3 relative to wild-type and its seeds contained 30.1 %DW more FA and 32.6 %DW more TAG than wild-type. Cell suspension cultures were generated from leaves of badc1badc3 and wild-type plants to test whether BADC contributes to the irreversible phase of ACCase inhibition resulting from culture in medium containing 10mM Tween80. While the reversible phase of ACCase inhibition after two days of Tween80 feeding was equivalent for badc1badc3 and wild-type, the irreversible phase of inhibition following four days of Tween80 feeding was reduced by 50% in badc1badc3 relative to wild-type. In this work we present evidence for two important homeostatic roles for BADC proteins in downregulating ACCase activity: during normal growth and development, and by contributing to its long-term irreversible feedback inhibition resulting from oversupply of fatty acids.« less

Biotin Attachment Domain-Containing Proteins Irreversibly Inhibit Acetyl CoA Carboxylase

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

Keereetaweep, Jantana; Liu, Hui; Zhai, Zhiyang

The first committed step in fatty acid synthesis is mediated by Acetyl-CoA carboxylase (ACCase), a biotin-dependent enzyme that carboxylates acetyl-CoA to produce malonyl-CoA. ACCase can be feedback-regulated by short-term (reversible) and longer-term (irreversible) inhibition upon oversupply of fatty acids (FA) provided by Tween80 (predominantly containing oleic acid; 18:1). Biotin-Attachment-Domain-Containing (BADC) proteins are inactive analogs of biotin carboxyl transfer protein (BCCP) that lack biotin and their incorporation into ACCase downregulates it by displacing active (biotin-containing) BCCP subunits. Individual T-DNA insertion lines of BADC1, BADC2, and BADC3 were used to generate badc1badc2 and badc1badc3. The badc1badc3 mutant and wild-type exhibited normal growthmore » and development, however ACCase activity was 26% higher in badc1badc3 relative to wild-type and its seeds contained 30.1 %DW more FA and 32.6 %DW more TAG than wild-type. Cell suspension cultures were generated from leaves of badc1badc3 and wild-type plants to test whether BADC contributes to the irreversible phase of ACCase inhibition resulting from culture in medium containing 10mM Tween80. While the reversible phase of ACCase inhibition after two days of Tween80 feeding was equivalent for badc1badc3 and wild-type, the irreversible phase of inhibition following four days of Tween80 feeding was reduced by 50% in badc1badc3 relative to wild-type. In this work we present evidence for two important homeostatic roles for BADC proteins in downregulating ACCase activity: during normal growth and development, and by contributing to its long-term irreversible feedback inhibition resulting from oversupply of fatty acids.« less

Evidence that the tandem-pleckstrin-homology-domain-containing protein TAPP1 interacts with Ptd(3,4)P2 and the multi-PDZ-domain-containing protein MUPP1 in vivo.