Self-Assembly in the Ferritin Nano-Cage Protein Superfamily

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Yu Zhang

2011-08-01

Full Text Available Protein self-assembly, through specific, high affinity, and geometrically constraining protein-protein interactions, can control and lead to complex cellular nano-structures. Establishing an understanding of the underlying principles that govern protein self-assembly is not only essential to appreciate the fundamental biological functions of these structures, but could also provide a basis for their enhancement for nano-material applications. The ferritins are a superfamily of well studied proteins that self-assemble into hollow cage-like structures which are ubiquitously found in both prokaryotes and eukaryotes. Structural studies have revealed that many members of the ferritin family can self-assemble into nano-cages of two types. Maxi-ferritins form hollow spheres with octahedral symmetry composed of twenty-four monomers. Mini-ferritins, on the other hand, are tetrahedrally symmetric, hollow assemblies composed of twelve monomers. This review will focus on the structure of members of the ferritin superfamily, the mechanism of ferritin self-assembly and the structure-function relations of these proteins.

Chlorpyrifos, chlorpyrifos-oxon, and diisopropylfluorophosphate inhibit kinesin-dependent microtubule motility

International Nuclear Information System (INIS)

Gearhart, Debra A.; Sickles, Dale W.; Buccafusco, Jerry J.; Prendergast, Mark A.; Terry, Alvin V.

2007-01-01

Diisopropylfluorophosphate, originally developed as a chemical warfare agent, is structurally similar to nerve agents, and chlorpyrifos has extensive worldwide use as an agricultural pesticide. While inhibition of cholinesterases underlies the acute toxicity of these organophosphates, we previously reported impaired axonal transport in the sciatic nerves from rats treated chronically with subthreshold doses of chlorpyrifos. Those data indicate that chlorpyrifos (and/or its active metabolite, chlorpyrifos-oxon) might directly affect the function of kinesin and/or microtubules-the principal proteins that mediate anterograde axonal transport. The current report describes in vitro assays to assess the concentration-dependent effects of chlorpyrifos (0-10 μM), chlorpyrifos-oxon (0-10 μM), and diisopropylfluorophosphate (0-0.59 nM) on kinesin-dependent microtubule motility. Preincubating bovine brain microtubules with the organophosphates did not alter kinesin-mediated microtubule motility. In contrast, preincubation of bovine brain kinesin with diisopropylfluorophosphate, chlorpyrifos, or chlorpyrifos-oxon produced a concentration-dependent increase in the number of locomoting microtubules that detached from the kinesin-coated glass cover slip. Our data suggest that the organophosphates-chlorpyrifos-oxon, chlorpyrifos, and diisopropylfluorophosphate-directly affect kinesin, thereby disrupting kinesin-dependent transport on microtubules. Kinesin-dependent movement of vesicles, organelles, and other cellular components along microtubules is fundamental to the organization of all eukaryotic cells, especially in neurons where organelles and proteins synthesized in the cell body must move down long axons to pre-synaptic sites in nerve terminals. We postulate that disruption of kinesin-dependent intracellular transport could account for some of the long-term effects of organophosphates on the peripheral and central nervous system

Xenopus laevis Kif18A is a highly processive kinesin required for meiotic spindle integrity

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Martin M. Möckel

2017-04-01

Full Text Available The assembly and functionality of the mitotic spindle depends on the coordinated activities of microtubule-associated motor proteins of the dynein and kinesin superfamily. Our current understanding of the function of motor proteins is significantly shaped by studies using Xenopus laevis egg extract as its open structure allows complex experimental manipulations hardly feasible in other model systems. Yet, the Kinesin-8 orthologue of human Kif18A has not been described in Xenopus laevis so far. Here, we report the cloning and characterization of Xenopus laevis (Xl Kif18A. Xenopus Kif18A is expressed during oocyte maturation and its depletion from meiotic egg extract results in severe spindle defects. These defects can be rescued by wild-type Kif18A, but not Kif18A lacking motor activity or the C-terminus. Single-molecule microscopy assays revealed that Xl_Kif18A possesses high processivity, which depends on an additional C-terminal microtubule-binding site. Human tissue culture cells depleted of endogenous Kif18A display mitotic defects, which can be rescued by wild-type, but not tail-less Xl_Kif18A. Thus, Xl_Kif18A is the functional orthologue of human Kif18A whose activity is essential for the correct function of meiotic spindles in Xenopus oocytes.

Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast

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Gergely, Zachary R.; Crapo, Ammon; Hough, Loren E.; McIntosh, J. Richard; Betterton, Meredith D.

2016-01-01

Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8–deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8–induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen. PMID:27146110

Kinesin-1 and mitochondrial motility control by discrimination of structurally equivalent but distinct subdomains in Ran-GTP-binding domains of Ran-binding protein 2.

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Patil, Hemangi; Cho, Kyoung-in; Lee, James; Yang, Yi; Orry, Andrew; Ferreira, Paulo A

2013-03-27

The pleckstrin homology (PH) domain is a versatile fold that mediates a variety of protein-protein and protein-phosphatidylinositol lipid interactions. The Ran-binding protein 2 (RanBP2) contains four interspersed Ran GTPase-binding domains (RBD(n = 1-4)) with close structural homology to the PH domain of Bruton's tyrosine kinase. The RBD2, kinesin-binding domain (KBD) and RBD3 comprise a tripartite domain (R2KR3) of RanBP2 that causes the unfolding, microtubule binding and biphasic activation of kinesin-1, a crucial anterograde motor of mitochondrial motility. However, the interplay between Ran GTPase and R2KR3 of RanBP2 in kinesin-1 activation and mitochondrial motility is elusive. We use structure-function, biochemical, kinetic and cell-based assays with time-lapse live-cell microscopy of over 260,000 mitochondrial-motility-related events to find mutually exclusive subdomains in RBD2 and RBD3 towards Ran GTPase binding, kinesin-1 activation and mitochondrial motility regulation. The RBD2 and RBD3 exhibit Ran-GTP-independent, subdomain and stereochemical-dependent discrimination on the biphasic kinetics of kinesin-1 activation or regulation of mitochondrial motility. Further, KBD alone and R2KR3 stimulate and suppress, respectively, multiple biophysical parameters of mitochondrial motility. The regulation of the bidirectional transport of mitochondria by either KBD or R2KR3 is highly coordinated, because their kinetic effects are accompanied always by changes in mitochondrial motile events of either transport polarity. These studies uncover novel roles in Ran GTPase-independent subdomains of RBD2 and RBD3, and KBD of RanBP2, that confer antagonizing and multi-modal mechanisms of kinesin-1 activation and regulation of mitochondrial motility. These findings open new venues towards the pharmacological harnessing of cooperative and competitive mechanisms regulating kinesins, RanBP2 or mitochondrial motility in disparate human disorders.

Structural analysis of intermolecular interactions in the kinesin adaptor complex fasciculation and elongation protein zeta 1/ short coiled-coil protein (FEZ1/SCOCO.

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Marcos Rodrigo Alborghetti

Full Text Available Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans, SCOCO (short coiled-coil protein / UNC-69 and kinesins (e.g. kinesin heavy chain / UNC116 are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth, we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance, cross-linking coupled with mass spectrometry (MS, SAXS (Small Angle X-ray Scattering and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance studies of the region involved in this process, corresponding to FEZ1 (92-194. Through studies involving the protein in its monomeric configuration (reduced and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.

Heterogeneity in kinesin function

NARCIS (Netherlands)

Reddy, Babu J N; Tripathy, Suvranta; Vershinin, Michael; Tanenbaum, Marvin E; Xu, Jing; Mattson-Hoss, Michelle; Arabi, Karim; Chapman, Dail; Doolin, Tory; Hyeon, Changbong; Gross, Steven P

2017-01-01

The kinesin family proteins are often studied as prototypical molecular motors; a deeper understanding of them can illuminate regulation of intracellular transport. It is typically assumed that they function identically. Here we find that this assumption of homogeneous function appears incorrect:

Tumour Suppressor Adenomatous Polyposis Coli (APC) localisation is regulated by both Kinesin-1 and Kinesin-2

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Ruane, Peter T; Gumy, Laura F; Bola, Becky; Anderson, Beverley; Wozniak, Marcin J; Hoogenraad, Casper C; Allan, Victoria J

2016-01-01

Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members,

The kinesin spindle protein inhibitor filanesib enhances the activity of pomalidomide and dexamethasone in multiple myeloma

OpenAIRE

Hernández-García, Susana; San-Segundo, Laura; González-Méndez, Lorena; Corchete, Luis A; Misiewicz-Krzeminska, Irena; Martín-Sánchez, Montserrat; López-Iglesias, Ana-Alicia; Algarín, Esperanza Macarena; Mogollón, Pedro; Díaz-Tejedor, Andrea; Paíno, Teresa; Tunquist, Brian; Mateos, María-Victoria; Gutiérrez, Norma C; Díaz-Rodriguez, Elena

2017-01-01

[EN]Kinesin spindle protein inhibition is known to be an effective therapeutic approach in several malignancies. Filanesib (ARRY-520), an inhibitor of this protein, has demonstrated activity in heavily pre-treated multiple myeloma patients. The aim of the work herein was to investigate the activity of filanesib in combination with pomalidomide plus dexamethasone backbone, and the mechanisms underlying the potential synergistic effect. The ability of filanesib to enhance the activity of pomali...

Dauer pheromone and G-protein signaling modulate the coordination of intraflagellar transport kinesin motor proteins in C. elegans

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J.A. Burghoorn (Jan); M.P.J. Dekkers (Martijn); S. Rademakers (Suzanne); A.A.W. de Jong (Ton); R. Willemsen (Rob); P. Swoboda (Peter); J. McCafferty (Gert)

2010-01-01

textabstractCilia length and function are dynamically regulated by modulation of intraflagellar transport (IFT). The cilia of C. elegans amphid channel neurons provide an excellent model to study this process, since they use two different kinesins for anterograde transport: kinesin-II and OSM-3

Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

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Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

2002-01-01

Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

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Marc Lenoir

2015-10-01

Full Text Available The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH and Tec homology (TH domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

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Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

2015-10-23

The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

Using sequence similarity networks for visualization of relationships across diverse protein superfamilies.

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Holly J Atkinson

Full Text Available The dramatic increase in heterogeneous types of biological data--in particular, the abundance of new protein sequences--requires fast and user-friendly methods for organizing this information in a way that enables functional inference. The most widely used strategy to link sequence or structure to function, homology-based function prediction, relies on the fundamental assumption that sequence or structural similarity implies functional similarity. New tools that extend this approach are still urgently needed to associate sequence data with biological information in ways that accommodate the real complexity of the problem, while being accessible to experimental as well as computational biologists. To address this, we have examined the application of sequence similarity networks for visualizing functional trends across protein superfamilies from the context of sequence similarity. Using three large groups of homologous proteins of varying types of structural and functional diversity--GPCRs and kinases from humans, and the crotonase superfamily of enzymes--we show that overlaying networks with orthogonal information is a powerful approach for observing functional themes and revealing outliers. In comparison to other primary methods, networks provide both a good representation of group-wise sequence similarity relationships and a strong visual and quantitative correlation with phylogenetic trees, while enabling analysis and visualization of much larger sets of sequences than trees or multiple sequence alignments can easily accommodate. We also define important limitations and caveats in the application of these networks. As a broadly accessible and effective tool for the exploration of protein superfamilies, sequence similarity networks show great potential for generating testable hypotheses about protein structure-function relationships.

Using sequence similarity networks for visualization of relationships across diverse protein superfamilies.

Science.gov (United States)

Atkinson, Holly J; Morris, John H; Ferrin, Thomas E; Babbitt, Patricia C

2009-01-01

The dramatic increase in heterogeneous types of biological data--in particular, the abundance of new protein sequences--requires fast and user-friendly methods for organizing this information in a way that enables functional inference. The most widely used strategy to link sequence or structure to function, homology-based function prediction, relies on the fundamental assumption that sequence or structural similarity implies functional similarity. New tools that extend this approach are still urgently needed to associate sequence data with biological information in ways that accommodate the real complexity of the problem, while being accessible to experimental as well as computational biologists. To address this, we have examined the application of sequence similarity networks for visualizing functional trends across protein superfamilies from the context of sequence similarity. Using three large groups of homologous proteins of varying types of structural and functional diversity--GPCRs and kinases from humans, and the crotonase superfamily of enzymes--we show that overlaying networks with orthogonal information is a powerful approach for observing functional themes and revealing outliers. In comparison to other primary methods, networks provide both a good representation of group-wise sequence similarity relationships and a strong visual and quantitative correlation with phylogenetic trees, while enabling analysis and visualization of much larger sets of sequences than trees or multiple sequence alignments can easily accommodate. We also define important limitations and caveats in the application of these networks. As a broadly accessible and effective tool for the exploration of protein superfamilies, sequence similarity networks show great potential for generating testable hypotheses about protein structure-function relationships.

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

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Heinz, Eva; Lithgow, Trevor

2014-01-01

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

SVM-Fold: a tool for discriminative multi-class protein fold and superfamily recognition.

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Melvin, Iain; Ie, Eugene; Kuang, Rui; Weston, Jason; Stafford, William Noble; Leslie, Christina

2007-05-22

Predicting a protein's structural class from its amino acid sequence is a fundamental problem in computational biology. Much recent work has focused on developing new representations for protein sequences, called string kernels, for use with support vector machine (SVM) classifiers. However, while some of these approaches exhibit state-of-the-art performance at the binary protein classification problem, i.e. discriminating between a particular protein class and all other classes, few of these studies have addressed the real problem of multi-class superfamily or fold recognition. Moreover, there are only limited software tools and systems for SVM-based protein classification available to the bioinformatics community. We present a new multi-class SVM-based protein fold and superfamily recognition system and web server called SVM-Fold, which can be found at http://svm-fold.c2b2.columbia.edu. Our system uses an efficient implementation of a state-of-the-art string kernel for sequence profiles, called the profile kernel, where the underlying feature representation is a histogram of inexact matching k-mer frequencies. We also employ a novel machine learning approach to solve the difficult multi-class problem of classifying a sequence of amino acids into one of many known protein structural classes. Binary one-vs-the-rest SVM classifiers that are trained to recognize individual structural classes yield prediction scores that are not comparable, so that standard "one-vs-all" classification fails to perform well. Moreover, SVMs for classes at different levels of the protein structural hierarchy may make useful predictions, but one-vs-all does not try to combine these multiple predictions. To deal with these problems, our method learns relative weights between one-vs-the-rest classifiers and encodes information about the protein structural hierarchy for multi-class prediction. In large-scale benchmark results based on the SCOP database, our code weighting approach

A histone-like protein of mycobacteria possesses ferritin superfamily protein-like activity and protects against DNA damage by Fenton reaction.

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Masaki Takatsuka

Full Text Available Iron is an essential metal for living organisms but its level must be strictly controlled in cells, because ferrous ion induces toxicity by generating highly active reactive oxygen, hydroxyl radicals, through the Fenton reaction. In addition, ferric ion shows low solubility under physiological conditions. To overcome these obstacles living organisms possess Ferritin superfamily proteins that are distributed in all three domains of life: bacteria, archaea, and eukaryotes. These proteins minimize hydroxyl radical formation by ferroxidase activity that converts Fe(2+ into Fe(3+ and sequesters iron by storing it as a mineral inside a protein cage. In this study, we discovered that mycobacterial DNA-binding protein 1 (MDP1, a histone-like protein, has similar activity to ferritin superfamily proteins. MDP1 prevented the Fenton reaction and protects DNA by the ferroxidase activity. The K(m values of the ferroxidase activity by MDP1 of Mycobacterium bovis bacillus Calmette-Guérin (BCG-3007c, Mycobacterium tuberculosis (Rv2986c, and Mycobacterium leprae (ML1683; ML-LBP were 0.292, 0.252, and 0.129 mM, respectively. Furthermore, one MDP1 molecule directly captured 81.4±19.1 iron atoms, suggesting the role of this protein in iron storage. This study describes for the first time a ferroxidase-iron storage protein outside of the ferritin superfamily proteins and the protective role of this bacterial protein from DNA damage.

Shaping the tracks : Regulation of microtubule dynamics by kinesins KIF21A and KIF21B

NARCIS (Netherlands)

van Riel, W.E.|info:eu-repo/dai/nl/338772634

2016-01-01

Control of microtubule dynamics is important for cell morphogenesis. Kinesins, motor proteins known to function in cargo transport, were recently also implicated in altering the microtubule network. Several kinesins are described to cause microtubule network reorganization or stabilization, either

FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry.

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Martin, Douglas S; Fathi, Reza; Mitchison, Timothy J; Gelles, Jeff

2010-03-23

As the smallest and simplest motor enzymes, kinesins have served as the prototype for understanding the relationship between protein structure and mechanochemical function of enzymes in this class. Conventional kinesin (kinesin-1) is a motor enzyme that transports cargo toward the plus end of microtubules by a processive, asymmetric hand-over-hand mechanism. The coiled-coil neck domain, which connects the two kinesin motor domains, contributes to kinesin processivity (the ability to take many steps in a row) and is proposed to be a key determinant of the asymmetry in the kinesin mechanism. While previous studies have defined the orientation and position of microtubule-bound kinesin motor domains, the disposition of the neck coiled-coil remains uncertain. We determined the neck coiled-coil orientation using a multidonor fluorescence resonance energy transfer (FRET) technique to measure distances between microtubules and bound kinesin molecules. Microtubules were labeled with a new fluorescent taxol donor, TAMRA-X-taxol, and kinesin derivatives with an acceptor fluorophore attached at positions on the motor and neck coiled-coil domains were used to reconstruct the positions and orientations of the domains. FRET measurements to positions on the motor domain were largely consistent with the domain orientation determined in previous studies, validating the technique. Measurements to positions on the neck coiled-coil were inconsistent with a radial orientation and instead demonstrated that the neck coiled-coil is parallel to the microtubule surface. The measured orientation provides a structural explanation for how neck surface residues enhance processivity and suggests a simple hypothesis for the origin of kinesin step asymmetry and "limping."

Mechanical splitting of microtubules into protofilament bundles by surface-bound kinesin-1.

Science.gov (United States)

VanDelinder, Virginia; Adams, Peter G; Bachand, George D

2016-12-21

The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continue to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation.

Proteomics computational analyses suggest that baculovirus GP64 superfamily proteins are class III penetrenes

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Garry Robert F

2008-02-01

Full Text Available Abstract Background Members of the Baculoviridae encode two types of proteins that mediate virus:cell membrane fusion and penetration into the host cell. Alignments of primary amino acid sequences indicate that baculovirus fusion proteins of group I nucleopolyhedroviruses (NPV form the GP64 superfamily. The structure of these viral penetrenes has not been determined. The GP64 superfamily includes the glycoprotein (GP encoded by members of the Thogotovirus genus of the Orthomyxoviridae. The entry proteins of other baculoviruses, group II NPV and granuloviruses, are class I penetrenes. Results Class III penetrenes encoded by members of the Rhabdoviridae and Herpesviridae have an internal fusion domain comprised of beta sheets, other beta sheet domains, an extended alpha helical domain, a membrane proximal stem domain and a carboxyl terminal anchor. Similar sequences and structural/functional motifs that characterize class III penetrenes are located collinearly in GP64 of group I baculoviruses and related glycoproteins encoded by thogotoviruses. Structural models based on a prototypic class III penetrene, vesicular stomatitis virus glycoprotein (VSV G, were established for Thogoto virus (THOV GP and Autographa california multiple NPV (AcMNPV GP64 demonstrating feasible cysteine linkages. Glycosylation sites in THOV GP and AcMNPV GP64 appear in similar model locations to the two glycosylation sites of VSV G. Conclusion These results suggest that proteins in the GP64 superfamily are class III penetrenes.

Protein 4.1, a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERM superfamily.

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Aleksander F Sikorski

2007-01-01

Full Text Available The review is focused on the domain structure and function of protein 4.1, one of the proteins belonging to the membrane skeleton. The protein 4.1 of the red blood cells (4.1R is a multifunctional protein that localizes to the membrane skeleton and stabilizes erythrocyte shape and membrane mechanical properties, such as deformability and stability, via lateral interactions with spectrin, actin, glycophorin C and protein p55. Protein 4.1 binding is modulated through the action of kinases and/or calmodulin-Ca2+. Non-erythroid cells express the 4.1R homologues: 4.1G (general type, 4.1B (brain type, and 4.1N (neuron type, and the whole group belongs to the protein 4.1 superfamily, which is characterized by the presence of a highly conserved FERM domain at the N-terminus of the molecule. Proteins 4.1R, 4.1G, 4.1N and 4.1B are encoded by different genes. Most of the 4.1 superfamily proteins also contain an actin-binding domain. To date, more than 40 members have been identified. They can be divided into five groups: protein 4.1 molecules, ERM proteins, talin-related molecules, protein tyrosine phosphatase (PTPH proteins and NBL4 proteins. We have focused our attention on the main, well known representatives of 4.1 superfamily and tried to choose the proteins which are close to 4.1R or which have distinct functions. 4.1 family proteins are not just linkers between the plasma membrane and membrane skeleton; they also play an important role in various processes. Some, such as focal adhesion kinase (FAK, non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells, play the role in cell adhesion. The other members control or take part in tumor suppression, regulation of cell cycle progression, inhibition of cell proliferation, downstream signaling of the glutamate receptors, and establishment of cell polarity; some are also involved in cell proliferation, cell motility, and/or cell-to-cell communication.

Evolutionary Pattern of N-Glycosylation Sequon Numbers  in Eukaryotic ABC Protein Superfamilies

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R. Shyama Prasad Rao

2010-02-01

Full Text Available Many proteins contain a large number of NXS/T sequences (where X is any amino acid except proline which are the potential sites of asparagine (N linked glycosylation. However, the patterns of occurrence of these N-glycosylation sequons in related proteins or groups of proteins and their underlying causes have largely been unexplored. We computed the actual and probabilistic occurrence of NXS/T sequons in ABC protein superfamilies from eight diverse eukaryotic organisms. The ABC proteins contained significantly higher NXS/T sequon numbers compared to respective genome-wide average, but the sequon density was significantly lower owing to the increase in protein size and decrease in sequon specific amino acids. However, mammalian ABC proteins have significantly higher sequon density, and both serine and threonine containing sequons (NXS and NXT have been positively selected—against the recent findings of only threonine specific Darwinian selection of sequons in proteins. The occurrence of sequons was positively correlated with the frequency of sequon specific amino acids and negatively correlated with proline and the NPS/T sequences. Further, the NPS/T sequences were significantly higher than expected in plant ABC proteins which have the lowest number of NXS/T sequons. Accord- ingly, compared to overall proteins, N-glycosylation sequons in ABC protein superfamilies have a distinct pattern of occurrence, and the results are discussed in an evolutionary perspective.

GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity.

Science.gov (United States)

Shagin, Dmitry A; Barsova, Ekaterina V; Yanushevich, Yurii G; Fradkov, Arkady F; Lukyanov, Konstantin A; Labas, Yulii A; Semenova, Tatiana N; Ugalde, Juan A; Meyers, Ann; Nunez, Jose M; Widder, Edith A; Lukyanov, Sergey A; Matz, Mikhail V

2004-05-01

Homologs of the green fluorescent protein (GFP), including the recently described GFP-like domains of certain extracellular matrix proteins in Bilaterian organisms, are remarkably similar at the protein structure level, yet they often perform totally unrelated functions, thereby warranting recognition as a superfamily. Here we describe diverse GFP-like proteins from previously undersampled and completely new sources, including hydromedusae and planktonic Copepoda. In hydromedusae, yellow and nonfluorescent purple proteins were found in addition to greens. Notably, the new yellow protein seems to follow exactly the same structural solution to achieving the yellow color of fluorescence as YFP, an engineered yellow-emitting mutant variant of GFP. The addition of these new sequences made it possible to resolve deep-level phylogenetic relationships within the superfamily. Fluorescence (most likely green) must have already existed in the common ancestor of Cnidaria and Bilateria, and therefore GFP-like proteins may be responsible for fluorescence and/or coloration in virtually any animal. At least 15 color diversification events can be inferred following the maximum parsimony principle in Cnidaria. Origination of red fluorescence and nonfluorescent purple-blue colors on several independent occasions provides a remarkable example of convergent evolution of complex features at the molecular level.

Kinesin expands and stabilizes the GDP-microtubule lattice

Science.gov (United States)

Peet, Daniel R.; Burroughs, Nigel J.; Cross, Robert A.

2018-05-01

Kinesin-1 is a nanoscale molecular motor that walks towards the fast-growing (plus) ends of microtubules, hauling molecular cargo to specific reaction sites in cells. Kinesin-driven transport is central to the self-organization of eukaryotic cells and shows great promise as a tool for nano-engineering1. Recent work hints that kinesin may also play a role in modulating the stability of its microtubule track, both in vitro2,3 and in vivo4, but the results are conflicting5-7 and the mechanisms are unclear. Here, we report a new dimension to the kinesin-microtubule interaction, whereby strong-binding state (adenosine triphosphate (ATP)-bound and apo) kinesin-1 motor domains inhibit the shrinkage of guanosine diphosphate (GDP) microtubules by up to two orders of magnitude and expand their lattice spacing by 1.6%. Our data reveal an unexpected mechanism by which the mechanochemical cycles of kinesin and tubulin interlock, and so allow motile kinesins to influence the structure, stability and mechanics of their microtubule track.

Phosphatidylcholine Transfer Protein Interacts with Thioesterase Superfamily Member 2 to Attenuate Insulin Signaling

OpenAIRE

Ersoy, Baran A.; Tarun, Akansha; D’Aquino, Katharine; Hancer, Nancy J.; Ukomadu, Chinweike; White, Morris F.; Michel, Thomas; Manning, Brendan D.; Cohen, David E.

2013-01-01

Phosphatidylcholine transfer protein (PC-TP) is a phospholipid-binding protein that is enriched in liver and that interacts with thioesterase superfamily member 2 (THEM2). Mice lacking either protein exhibit improved hepatic glucose homeostasis and are resistant to diet-induced diabetes. Insulin receptor substrate 2 (IRS2) and mammalian target of rapamycin complex 1 (mTORC1) are key effectors of insulin signaling, which is attenuated in diabetes. We found that PC-TP inhibited IRS2, as evidenc...

Activity of the kinesin spindle protein inhibitor ispinesib (SB-715992) in models of breast cancer

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Purcell, James W; Davis, Jefferson; Reddy, Mamatha; Martin, Shamra; Samayoa, Kimberly; Vo, Hung; Thomsen, Karen; Bean, Peter; Kuo, Wen Lin; Ziyad, Safiyyah; Billig, Jessica; Feiler, Heidi S; Gray, Joe W; Wood, Kenneth W; Cases, Sylvaine

2009-06-10

Ispinesib (SB-715992) is a potent inhibitor of kinesin spindle protein (KSP), a kinesin motor protein essential for the formation of a bipolar mitotic spindle and cell cycle progression through mitosis. Clinical studies of ispinesib have demonstrated a 9% response rate in patients with locally advanced or metastatic breast cancer, and a favorable safety profile without significant neurotoxicities, gastrointestinal toxicities or hair loss. To better understand the potential of ispinesib in the treatment of breast cancer we explored the activity of ispinesib alone and in combination several therapies approved for the treatment of breast cancer. We measured the ispinesib sensitivity and pharmacodynamic response of breast cancer cell lines representative of various subtypes in vitro and as xenografts in vivo, and tested the ability of ispinesib to enhance the anti-tumor activity of approved therapies. In vitro, ispinesib displayed broad anti-proliferative activity against a panel of 53 breast cell-lines. In vivo, ispinesib produced regressions in each of five breast cancer models, and tumor free survivors in three of these models. The effects of ispinesib treatment on pharmacodynamic markers of mitosis and apoptosis were examined in vitro and in vivo, revealing a greater increase in both mitotic and apoptotic markers in the MDA-MB-468 model than in the less sensitive BT-474 model. In vivo, ispinesib enhanced the anti-tumor activity of trastuzumab, lapatinib, doxorubicin, and capecitabine, and exhibited activity comparable to paclitaxel and ixabepilone. These findings support further clinical exploration of KSP inhibitors for the treatment of breast cancer.

The actin-binding protein capulet genetically interacts with the microtubule motor kinesin to maintain neuronal dendrite homeostasis.

Directory of Open Access Journals (Sweden)

Paul M B Medina

Full Text Available BACKGROUND: Neurons require precise cytoskeletal regulation within neurites, containing microtubule tracks for cargo transport in axons and dendrites or within synapses containing organized actin. Due to the unique architecture and specialized function of neurons, neurons are particularly susceptible to perturbation of the cytoskeleton. Numerous actin-binding proteins help maintain proper cytoskeletal regulation. METHODOLOGY/PRINCIPAL FINDINGS: From a Drosophila forward genetic screen, we identified a mutation in capulet--encoding a conserved actin-binding protein--that causes abnormal aggregates of actin within dendrites. Through interaction studies, we demonstrate that simultaneous genetic inactivation of capulet and kinesin heavy chain, a microtubule motor protein, produces elongate cofilin-actin rods within dendrites but not axons. These rods resemble actin-rich structures induced in both mammalian neurodegenerative and Drosophila Alzheimer's models, but have not previously been identified by loss of function mutations in vivo. We further demonstrate that mitochondria, which are transported by Kinesin, have impaired distribution along dendrites in a capulet mutant. While Capulet and Cofilin may biochemically cooperate in certain circumstances, in neuronal dendrites they genetically antagonize each other. CONCLUSIONS/SIGNIFICANCE: The present study is the first molecularly defined loss of function demonstration of actin-cofilin rods in vivo. This study suggests that simultaneous, seemingly minor perturbations in neuronal dendrites can synergize producing severe abnormalities affecting actin, microtubules and mitochondria/energy availability in dendrites. Additionally, as >90% of Alzheimer's and Parkinson's cases are sporadic this study suggests mechanisms by which multiple mutations together may contribute to neurodegeneration instead of reliance on single mutations to produce disease.

Mutation of Rice BC12/GDD1, Which Encodes a Kinesin-Like Protein That Binds to a GA Biosynthesis Gene Promoter, Leads to Dwarfism with Impaired Cell Elongation[W][OA

Science.gov (United States)

Li, Juan; Jiang, Jiafu; Qian, Qian; Xu, Yunyuan; Zhang, Cui; Xiao, Jun; Du, Cheng; Luo, Wei; Zou, Guoxing; Chen, Mingluan; Huang, Yunqing; Feng, Yuqi; Cheng, Zhukuan; Yuan, Ming; Chong, Kang

2011-01-01

The kinesins are a family of microtubule-based motor proteins that move directionally along microtubules and are involved in many crucial cellular processes, including cell elongation in plants. Less is known about kinesins directly regulating gene transcription to affect cellular physiological processes. Here, we describe a rice (Oryza sativa) mutant, gibberellin-deficient dwarf1 (gdd1), that has a phenotype of greatly reduced length of root, stems, spikes, and seeds. This reduced length is due to decreased cell elongation and can be rescued by exogenous gibberellic acid (GA3) treatment. GDD1 was cloned by a map-based approach, was expressed constitutively, and was found to encode the kinesin-like protein BRITTLE CULM12 (BC12). Microtubule cosedimentation assays revealed that BC12/GDD1 bound to microtubules in an ATP-dependent manner. Whole-genome microarray analysis revealed the expression of ent-kaurene oxidase (KO2), which encodes an enzyme involved in GA biosynthesis, was downregulated in gdd1. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that GDD1 bound to the element ACCAACTTGAA in the KO2 promoter. In addition, GDD1 was shown to have transactivation activity. The level of endogenous GAs was reduced in gdd1, and the reorganization of cortical microtubules was altered. Therefore, BC12/GDD1, a kinesin-like protein with transcription regulation activity, mediates cell elongation by regulating the GA biosynthesis pathway in rice. PMID:21325138

Kinesin-1 plays a role in transport of SNAP-25 to the plasma membrane

Energy Technology Data Exchange (ETDEWEB)

Morton, April M.; Cunningham, Anthony L. [Centre for Virus Research, Westmead Millennium Institute, The University of Sydney and Westmead Hospital, Westmead, NSW 2145 (Australia); Diefenbach, Russell J., E-mail: russell_diefenbach@wmi.usyd.edu.au [Centre for Virus Research, Westmead Millennium Institute, The University of Sydney and Westmead Hospital, Westmead, NSW 2145 (Australia)

2010-01-01

The cellular molecular motor kinesin-1 mediates the microtubule-dependent transport of a range of cargo. We have previously identified an interaction between the cargo-binding domain of kinesin-1 heavy chain KIF5B and the membrane-associated SNARE proteins SNAP-25 and SNAP-23. In this study we further defined the minimal SNAP-25 binding domain in KIF5B to residues 874-894. Overexpression of a fragment of KIF5B (residues 594-910) resulted in significant colocalization with SNAP-25 with resulting blockage of the trafficking of SNAP-25 to the periphery of cells. This indicates that kinesin-1 facilitates the transport of SNAP-25 containing vesicles as a prerequisite to SNAP-25 driven membrane fusion events.

Kinesin and Dynein Mechanics: Measurement Methods and Research Applications.

Science.gov (United States)

Abraham, Zachary; Hawley, Emma; Hayosh, Daniel; Webster-Wood, Victoria A; Akkus, Ozan

2018-02-01

Motor proteins play critical roles in the normal function of cells and proper development of organisms. Among motor proteins, failings in the normal function of two types of proteins, kinesin and dynein, have been shown to lead many pathologies, including neurodegenerative diseases and cancers. As such, it is critical to researchers to understand the underlying mechanics and behaviors of these proteins, not only to shed light on how failures may lead to disease, but also to guide research toward novel treatment and nano-engineering solutions. To this end, many experimental techniques have been developed to measure the force and motility capabilities of these proteins. This review will (a) discuss such techniques, specifically microscopy, atomic force microscopy (AFM), optical trapping, and magnetic tweezers, and (b) the resulting nanomechanical properties of motor protein functions such as stalling force, velocity, and dependence on adenosine triphosophate (ATP) concentrations will be comparatively discussed. Additionally, this review will highlight the clinical importance of these proteins. Furthermore, as the understanding of the structure and function of motor proteins improves, novel applications are emerging in the field. Specifically, researchers have begun to modify the structure of existing proteins, thereby engineering novel elements to alter and improve native motor protein function, or even allow the motor proteins to perform entirely new tasks as parts of nanomachines. Kinesin and dynein are vital elements for the proper function of cells. While many exciting experiments have shed light on their function, mechanics, and applications, additional research is needed to completely understand their behavior.

The mechanochemical cycle of mammalian kinesin-2 KIF3A/B under load

Science.gov (United States)

Andreasson, Johan O.L.; Shastry, Shankar; Hancock, William O.; Block, Steven M.

2015-01-01

Summary The response of motor proteins to external loads underlies their ability to work in teams and determines the net speed and directionality of cargo transport. The mammalian kinesin-2, KIF3A/B, is a heterotrimeric motor involved in intraflagellar transport and vesicle motility in neurons. Bidirectional cargo transport is known to result from the opposing activities of KIF3A/B and dynein bound to the same cargo, but the load-dependent properties of kinesin-2 are poorly understood. We used a feedback-controlled optical trap to probe the velocity, run length and unbinding kinetics of mouse KIF3A/B under various loads and nucleotide conditions. The kinesin-2 motor velocity is less sensitive than kinesin-1 to external forces, but its processivity diminishes steeply with load, and the motor was observed occasionally to slip and reattach. Each motor domain was characterized by studying homodimeric constructs, and a global fit to the data resulted in a comprehensive pathway that quantifies the principal force-dependent kinetic transitions. The properties of the KIF3A/B heterodimer are intermediate between the two homodimers, and the distinct load-dependent behavior is attributable to the properties of the motor domains, and not to the neck-linkers or the coiled-coil stalk. We conclude that the force-dependent movement of KIF3A/B differs significantly from conventional kinesin-1. Against opposing dynein forces, KIF3A/B motors are predicted to rapidly unbind and rebind, resulting in qualitatively different transport behavior from kinesin-1. PMID:25866395

Exploring and Expanding the Fatty-Acid-Binding Protein Superfamily in Fasciola Species.

Science.gov (United States)

Morphew, Russell M; Wilkinson, Toby J; Mackintosh, Neil; Jahndel, Veronika; Paterson, Steve; McVeigh, Paul; Abbas Abidi, Syed M; Saifullah, Khalid; Raman, Muthusamy; Ravikumar, Gopalakrishnan; LaCourse, James; Maule, Aaron; Brophy, Peter M

2016-09-02

The liver flukes Fasciola hepatica and F. gigantica infect livestock worldwide and threaten food security with climate change and problematic control measures spreading disease. Fascioliasis is also a foodborne disease with up to 17 million humans infected. In the absence of vaccines, treatment depends on triclabendazole (TCBZ), and overuse has led to widespread resistance, compromising future TCBZ control. Reductionist biology from many laboratories has predicted new therapeutic targets. To this end, the fatty-acid-binding protein (FABP) superfamily has proposed multifunctional roles, including functions intersecting vaccine and drug therapy, such as immune modulation and anthelmintic sequestration. Research is hindered by a lack of understanding of the full FABP superfamily complement. Although discovery studies predicted FABPs as promising vaccine candidates, it is unclear if uncharacterized FABPs are more relevant for vaccine formulations. We have coupled genome, transcriptome, and EST data mining with proteomics and phylogenetics to reveal a liver fluke FABP superfamily of seven clades: previously identified clades I-III and newly identified clades IV-VII. All new clade FABPs were analyzed using bioinformatics and cloned from both liver flukes. The extended FABP data set will provide new study tools to research the role of FABPs in parasite biology and as therapy targets.

Effects of Obstacles on the Dynamics of Kinesins, Including Velocity and Run Length, Predicted by a Model of Two Dimensional Motion.

Directory of Open Access Journals (Sweden)

Woochul Nam

Full Text Available Kinesins are molecular motors which walk along microtubules by moving their heads to different binding sites. The motion of kinesin is realized by a conformational change in the structure of the kinesin molecule and by a diffusion of one of its two heads. In this study, a novel model is developed to account for the 2D diffusion of kinesin heads to several neighboring binding sites (near the surface of microtubules. To determine the direction of the next step of a kinesin molecule, this model considers the extension in the neck linkers of kinesin and the dynamic behavior of the coiled-coil structure of the kinesin neck. Also, the mechanical interference between kinesins and obstacles anchored on the microtubules is characterized. The model predicts that both the kinesin velocity and run length (i.e., the walking distance before detaching from the microtubule are reduced by static obstacles. The run length is decreased more significantly by static obstacles than the velocity. Moreover, our model is able to predict the motion of kinesin when other (several motors also move along the same microtubule. Furthermore, it suggests that the effect of mechanical interaction/interference between motors is much weaker than the effect of static obstacles. Our newly developed model can be used to address unanswered questions regarding degraded transport caused by the presence of excessive tau proteins on microtubules.

Distribution of tubulin, kinesin, and dynein in light- and dark-adapted octopus retinas.

Science.gov (United States)

Martinez, J M; Elfarissi, H; De Velasco, B; Ochoa, G H; Miller, A M; Clark, Y M; Matsumoto, B; Robles, L J

2000-01-01

Cephalopod retinas exhibit several responses to light and dark adaptation, including rhabdom size changes, photopigment movements, and pigment granule migration. Light- and dark-directed rearrangements of microfilament and microtubule cytoskeletal transport pathways could drive these changes. Recently, we localized actin-binding proteins in light-/dark-adapted octopus rhabdoms and suggested that actin cytoskeletal rearrangements bring about the formation and degradation of rhabdomere microvilli subsets. To determine if the microtubule cytoskeleton and associated motor proteins control the other light/dark changes, we used immunoblotting and immunocytochemical procedures to map the distribution of tubulin, kinesin, and dynein in dorsal and ventral halves of light- and dark-adapted octopus retinas. Immunoblots detected alpha- and beta-tubulin, dynein intermediate chain, and kinesin heavy chain in extracts of whole retinas. Epifluorescence and confocal microscopy showed that the tubulin proteins were distributed throughout the retina with more immunoreactivity in retinas exposed to light. Kinesin localization was heavy in the pigment layer of light- and dark-adapted ventral retinas but was less prominent in the dorsal region. Dynein distribution also varied in dorsal and ventral retinas with more immunoreactivity in light- and dark-adapted ventral retinas and confocal microscopy emphasized the granular nature of this labeling. We suggest that light may regulate the distribution of microtubule cytoskeletal proteins in the octopus retina and that position, dorsal versus ventral, also influences the distribution of motor proteins. The microtubule cytoskeleton is most likely involved in pigment granule migration in the light and dark and with the movement of transport vesicles from the photoreceptor inner segments to the rhabdoms.

Kinesin-dependent mechanism for controlling triglyceride secretion from the liver.

Science.gov (United States)

Rai, Priyanka; Kumar, Mukesh; Sharma, Geetika; Barak, Pradeep; Das, Saumitra; Kamat, Siddhesh S; Mallik, Roop

2017-12-05

Despite massive fluctuations in its internal triglyceride content, the liver secretes triglyceride under tight homeostatic control. This buffering function is most visible after fasting, when liver triglyceride increases manyfold but circulating serum triglyceride barely fluctuates. How the liver controls triglyceride secretion is unknown, but is fundamentally important for lipid and energy homeostasis in animals. Here we find an unexpected cellular and molecular mechanism behind such control. We show that kinesin motors are recruited to triglyceride-rich lipid droplets (LDs) in the liver by the GTPase ARF1, which is a key activator of lipolysis. This recruitment is activated by an insulin-dependent pathway and therefore responds to fed/fasted states of the animal. In fed state, ARF1 and kinesin appear on LDs, consequently transporting LDs to the periphery of hepatocytes where the smooth endoplasmic reticulum (sER) is present. Because the lipases that catabolize LDs in hepatocytes reside on the sER, LDs can now be catabolized efficiently to provide triglyceride for lipoprotein assembly and secretion from the sER. Upon fasting, insulin is lowered to remove ARF1 and kinesin from LDs, thus down-regulating LD transport and sER-LD contacts. This tempers triglyceride availabiity for very low density lipoprotein assembly and allows homeostatic control of serum triglyceride in a fasted state. We further show that kinesin knockdown inhibits hepatitis-C virus replication in hepatocytes, likely because translated viral proteins are unable to transfer from the ER to LDs. Copyright © 2017 the Author(s). Published by PNAS.

The TULIP superfamily of eukaryotic lipid-binding proteins as a mediator of lipid sensing and transport.

Science.gov (United States)

Alva, Vikram; Lupas, Andrei N

2016-08-01

The tubular lipid-binding (TULIP) superfamily has emerged in recent years as a major mediator of lipid sensing and transport in eukaryotes. It currently encompasses three protein families, SMP-like, BPI-like, and Takeout-like, which share a common fold. This fold consists of a long helix wrapped in a highly curved anti-parallel β-sheet, enclosing a central, lipophilic cavity. The SMP-like proteins, which include subunits of the ERMES complex and the extended synaptotagmins (E-Syts), appear to be mainly located at membrane contacts sites (MCSs) between organelles, mediating inter-organelle lipid exchange. The BPI-like proteins, which include the bactericidal/permeability-increasing protein (BPI), the LPS (lipopolysaccharide)-binding protein (LBP), the cholesteryl ester transfer protein (CETP), and the phospholipid transfer protein (PLTP), are either involved in innate immunity against bacteria through their ability to sense lipopolysaccharides, as is the case for BPI and LBP, or in lipid exchange between lipoprotein particles, as is the case for CETP and PLTP. The Takeout-like proteins, which are comprised of insect juvenile hormone-binding proteins and arthropod allergens, transport, where known, lipid hormones to target tissues during insect development. In all cases, the activity of these proteins is underpinned by their ability to bind large, hydrophobic ligands in their central cavity and segregate them away from the aqueous environment. Furthermore, where they are involved in lipid exchange, recent structural studies have highlighted their ability to establish lipophilic, tubular channels, either between organelles in the case of SMP domains or between lipoprotein particles in the case of CETP. Here, we review the current knowledge on the structure, versatile functions, and evolution of the TULIP superfamily. We propose a deep evolutionary split in this superfamily, predating the Last Eukaryotic Common Ancestor, between the SMP-like proteins, which act on

Reversible control of kinesin activity and microtubule gliding speeds by switching the doping states of a conducting polymer support

Energy Technology Data Exchange (ETDEWEB)

Martin, Brett D [US Naval Research Laboratory, Code 6930, Washington, DC 20375 (United States); Velea, Luminita M [US Naval Research Laboratory, Code 6930, Washington, DC 20375 (United States); Soto, Carissa M [US Naval Research Laboratory, Code 6930, Washington, DC 20375 (United States); Whitaker, Craig M [US Naval Academy, Department of Chemistry, Annapolis, MD 21402 (United States); Gaber, Bruce P [US Naval Research Laboratory, Code 6930, Washington, DC 20375 (United States); Ratna, Banahalli [US Naval Research Laboratory, Code 6930, Washington, DC 20375 (United States)

2007-02-07

We describe a method for reversibly controlling the ATPase activity of streptavidin-linked kinesin by changing the doping states of a conducting polymer support. When the polymer (poly(CH{sub 2}OH-EDOT)) was electrochemically switched from its dedoped (semiconducting) state to its doped (conducting) state, the ATPase activity of the adsorbed kinesin complex decreased by 35% with a concomitant decrease in the gliding speeds of kinesin-driven microtubules. When the polymer was switched back to its original dedoped state, nearly identical increases were observed in the kinesin ATPase activity and microtubule speeds. Use of a fluorescent ATP substrate analogue showed that the total amount of kinesin adsorbed on the poly(CH{sub 2}OH-EDOT) surface remained constant as the doping state of the polymer was switched. The microtubules exhibited nearly identical speed differences on the doped and dedoped surfaces for both chemical and electrochemical doping methods. Michaelis-Menten modelling suggests that the doped surface acts as an 'uncompetitive inhibitor' of kinesin. This work represents an investigation into the phenomenon of an electrically switchable surface exerting a moderating effect on the activity of an adsorbed protein that does not contain a bound, electroactive metal ion.

Structure of TTHA1623, a novel metallo-β-lactamase superfamily protein from Thermus thermophilus HB8

International Nuclear Information System (INIS)

Yamamura, Akihiro; Okada, Akitoshi; Kameda, Yasuhiro; Ohtsuka, Jun; Nakagawa, Noriko; Ebihara, Akio; Nagata, Koji; Tanokura, Masaru

2009-01-01

The crystal structures of TTHA1623 from T. thermophilus HB8 in an iron-bound and a zinc-bound form have been determined to 2.8 and 2.2 Å resolution, respectively. TTHA1623 is a metallo-β-lactamase superfamily protein from the extremely thermophilic bacterium Thermus thermophilus HB8. Homologues of TTHA1623 exist in a wide range of bacteria and archaea and one eukaryote, Giardia lamblia, but their function remains unknown. To analyze the structural properties of TTHA1623, the crystal structures of its iron-bound and zinc-bound forms have been determined to 2.8 and 2.2 Å resolution, respectively. TTHA1623 possesses an αββα-fold similar to that of other metallo-β-lactamase superfamily proteins with glyoxalase II-type metal coordination. However, TTHA1623 exhibits a putative substrate-binding pocket with a unique shape

Radical SAM, A Novel Protein Superfamily Linking Unresolved Steps in Familiar Biosynthetic Pathways with Radical Mechanisms: Functional Characterization Using New Analysis and Information Visualization Methods

Energy Technology Data Exchange (ETDEWEB)

Sofia, Heidi J.; Chen, Guang; Hetzler, Elizabeth G.; Reyes Spindola, Jorge F.; Miller, Nancy E.

2001-03-01

A large protein superfamily with over 500 members has been discovered and analyzed using powerful new bioinformatics and information visualization methods. Evidence exists that these proteins generate a 5?-deoxyadenosyl radical by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. Radical SAM superfamily proteins function in DNA precursor, vitamin, cofactor, antibiotic, and herbicide biosynthesis in a collection of basic and familiar pathways. One of the members is interferon-inducible and is considered a candidate drug target for osteoporosis. The identification of this superfamily suggests that radical-based catalysis is important in a number of previously well-studied but unresolved biochemical pathways.

Highly loaded behavior of kinesins increases the robustness of transport under high resisting loads.

Directory of Open Access Journals (Sweden)

Woochul Nam

2015-03-01

Full Text Available Kinesins are nano-sized biological motors which walk by repeating a mechanochemical cycle. A single kinesin molecule is able to transport its cargo about 1 μm in the absence of external loads. However, kinesins perform much longer range transport in cells by working collectively. This long range of transport by a team of kinesins is surprising because the motion of the cargo in cells can be hindered by other particles. To reveal how the kinesins are able to accomplish their tasks of transport in harsh intracellular circumstances, stochastic studies on the kinesin motion are performed by considering the binding and unbinding of kinesins to microtubules and their dependence on the force acting on kinesin molecules. The unbinding probabilities corresponding to each mechanochemical state of kinesin are modeled. The statistical characterization of the instants and locations of binding are captured by computing the probability of unbound kinesin being at given locations. It is predicted that a group of kinesins has a more efficient transport than a single kinesin from the perspective of velocity and run length. Particularly, when large loads are applied, the leading kinesin remains bound to the microtubule for long time which increases the chances of the other kinesins to bind to the microtubule. To predict effects of this behavior of the leading kinesin under large loads on the collective transport, the motion of the cargo is studied when the cargo confronts obstacles. The result suggests that the behavior of kinesins under large loads prevents the early termination of the transport which can be caused by the interference with the static or moving obstacles.

Phylogenomic analysis of the GIY-YIG nuclease superfamily

Directory of Open Access Journals (Sweden)

Bujnicki Janusz M

2006-04-01

Full Text Available Abstract Background The GIY-YIG domain was initially identified in homing endonucleases and later in other selfish mobile genetic elements (including restriction enzymes and non-LTR retrotransposons and in enzymes involved in DNA repair and recombination. However, to date no systematic search for novel members of the GIY-YIG superfamily or comparative analysis of these enzymes has been reported. Results We carried out database searches to identify all members of known GIY-YIG nuclease families. Multiple sequence alignments together with predicted secondary structures of identified families were represented as Hidden Markov Models (HMM and compared by the HHsearch method to the uncharacterized protein families gathered in the COG, KOG, and PFAM databases. This analysis allowed for extending the GIY-YIG superfamily to include members of COG3680 and a number of proteins not classified in COGs and to predict that these proteins may function as nucleases, potentially involved in DNA recombination and/or repair. Finally, all old and new members of the GIY-YIG superfamily were compared and analyzed to infer the phylogenetic tree. Conclusion An evolutionary classification of the GIY-YIG superfamily is presented for the very first time, along with the structural annotation of all (subfamilies. It provides a comprehensive picture of sequence-structure-function relationships in this superfamily of nucleases, which will help to design experiments to study the mechanism of action of known members (especially the uncharacterized ones and will facilitate the prediction of function for the newly discovered ones.

Loading direction regulates the affinity of ADP for kinesin.

Science.gov (United States)

Uemura, Sotaro; Ishiwata, Shin'ichi

2003-04-01

Kinesin is an ATP-driven molecular motor that moves processively along a microtubule. Processivity has been explained as a mechanism that involves alternating single- and double-headed binding of kinesin to microtubules coupled to the ATPase cycle of the motor. The internal load imposed between the two bound heads has been proposed to be a key factor regulating the ATPase cycle in each head. Here we show that external load imposed along the direction of motility on a single kinesin molecule enhances the binding affinity of ADP for kinesin, whereas an external load imposed against the direction of motility decreases it. This coupling between loading direction and enzymatic activity is in accord with the idea that the internal load plays a key role in the unidirectional and cooperative movement of processive motors.

Combining protein sequence, structure, and dynamics: A novel approach for functional evolution analysis of PAS domain superfamily.

Science.gov (United States)

Dong, Zheng; Zhou, Hongyu; Tao, Peng

2018-02-01

PAS domains are widespread in archaea, bacteria, and eukaryota, and play important roles in various functions. In this study, we aim to explore functional evolutionary relationship among proteins in the PAS domain superfamily in view of the sequence-structure-dynamics-function relationship. We collected protein sequences and crystal structure data from RCSB Protein Data Bank of the PAS domain superfamily belonging to three biological functions (nucleotide binding, photoreceptor activity, and transferase activity). Protein sequences were aligned and then used to select sequence-conserved residues and build phylogenetic tree. Three-dimensional structure alignment was also applied to obtain structure-conserved residues. The protein dynamics were analyzed using elastic network model (ENM) and validated by molecular dynamics (MD) simulation. The result showed that the proteins with same function could be grouped by sequence similarity, and proteins in different functional groups displayed statistically significant difference in their vibrational patterns. Interestingly, in all three functional groups, conserved amino acid residues identified by sequence and structure conservation analysis generally have a lower fluctuation than other residues. In addition, the fluctuation of conserved residues in each biological function group was strongly correlated with the corresponding biological function. This research suggested a direct connection in which the protein sequences were related to various functions through structural dynamics. This is a new attempt to delineate functional evolution of proteins using the integrated information of sequence, structure, and dynamics. © 2017 The Protein Society.

New insights into potential functions for the protein 4.1superfamily of proteins in kidney epithelium

Energy Technology Data Exchange (ETDEWEB)

Calinisan, Venice; Gravem, Dana; Chen, Ray Ping-Hsu; Brittin,Sachi; Mohandas, Narla; Lecomte, Marie-Christine; Gascard, Philippe

2005-06-17

Members of the protein 4.1 family of adapter proteins are expressed in a broad panel of tissues including various epithelia where they likely play an important role in maintenance of cell architecture and polarity and in control of cell proliferation. We have recently characterized the structure and distribution of three members of the protein 4.1 family, 4.1B, 4.1R and 4.1N, in mouse kidney. We describe here binding partners for renal 4.1 proteins, identified through the screening of a rat kidney yeast two-hybrid system cDNA library. The identification of putative protein 4.1-based complexes enables us to envision potential functions for 4.1 proteins in kidney: organization of signaling complexes, response to osmotic stress, protein trafficking, and control of cell proliferation. We discuss the relevance of these protein 4.1-based interactions in kidney physio-pathology in the context of their previously identified functions in other cells and tissues. Specifically, we will focus on renal 4.1 protein interactions with beta amyloid precursor protein (beta-APP), 14-3-3 proteins, and the cell swelling-activated chloride channel pICln. We also discuss the functional relevance of another member of the protein 4.1 superfamily, ezrin, in kidney physiopathology.

Inference of functional properties from large-scale analysis of enzyme superfamilies.

Science.gov (United States)

Brown, Shoshana D; Babbitt, Patricia C

2012-01-02

As increasingly large amounts of data from genome and other sequencing projects become available, new approaches are needed to determine the functions of the proteins these genes encode. We show how large-scale computational analysis can help to address this challenge by linking functional information to sequence and structural similarities using protein similarity networks. Network analyses using three functionally diverse enzyme superfamilies illustrate the use of these approaches for facile updating and comparison of available structures for a large superfamily, for creation of functional hypotheses for metagenomic sequences, and to summarize the limits of our functional knowledge about even well studied superfamilies.

Systematic classification of the His-Me finger superfamily.

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Jablonska, Jagoda; Matelska, Dorota; Steczkiewicz, Kamil; Ginalski, Krzysztof

2017-11-16

The His-Me finger endonucleases, also known as HNH or ββα-metal endonucleases, form a large and diverse protein superfamily. The His-Me finger domain can be found in proteins that play an essential role in cells, including genome maintenance, intron homing, host defense and target offense. Its overall structural compactness and non-specificity make it a perfectly-tailored pathogenic module that participates on both sides of inter- and intra-organismal competition. An extremely low sequence similarity across the superfamily makes it difficult to identify and classify new His-Me fingers. Using state-of-the-art distant homology detection methods, we provide an updated and systematic classification of His-Me finger proteins. In this work, we identified over 100 000 proteins and clustered them into 38 groups, of which three groups are new and cannot be found in any existing public domain database of protein families. Based on an analysis of sequences, structures, domain architectures, and genomic contexts, we provide a careful functional annotation of the poorly characterized members of this superfamily. Our results may inspire further experimental investigations that should address the predicted activity and clarify the potential substrates, to provide more detailed insights into the fundamental biological roles of these proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

The aldo-keto reductase superfamily homepage.

Science.gov (United States)

Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

2003-02-01

The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

BRICHOS - a superfamily of multidomain proteins with diverse functions

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Johansson Jan

2009-09-01

Full Text Available Abstract Background The BRICHOS domain has been found in 8 protein families with a wide range of functions and a variety of disease associations, such as respiratory distress syndrome, dementia and cancer. The domain itself is thought to have a chaperone function, and indeed three of the families are associated with amyloid formation, but its structure and many of its functional properties are still unknown. Findings The proteins in the BRICHOS superfamily have four regions with distinct properties. We have analysed the BRICHOS proteins focusing on sequence conservation, amino acid residue properties, native disorder and secondary structure predictions. Residue conservation shows large variations between the regions, and the spread of residue conservation between different families can vary greatly within the regions. The secondary structure predictions for the BRICHOS proteins show remarkable coherence even where sequence conservation is low, and there seems to be little native disorder. Conclusions The greatly variant rates of conservation indicates different functional constraints among the regions and among the families. We present three previously unknown BRICHOS families; group A, which may be ancestral to the ITM2 families; group B, which is a close relative to the gastrokine families, and group C, which appears to be a truly novel, disjoint BRICHOS family. The C-terminal region of group C has nearly identical sequences in all species ranging from fish to man and is seemingly unique to this family, indicating critical functional or structural properties.

Inference of Functional Properties from Large-scale Analysis of Enzyme Superfamilies*

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Brown, Shoshana D.; Babbitt, Patricia C.

2012-01-01

As increasingly large amounts of data from genome and other sequencing projects become available, new approaches are needed to determine the functions of the proteins these genes encode. We show how large-scale computational analysis can help to address this challenge by linking functional information to sequence and structural similarities using protein similarity networks. Network analyses using three functionally diverse enzyme superfamilies illustrate the use of these approaches for facile updating and comparison of available structures for a large superfamily, for creation of functional hypotheses for metagenomic sequences, and to summarize the limits of our functional knowledge about even well studied superfamilies. PMID:22069325

Mapping the structural and dynamical features of kinesin motor domains.

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Guido Scarabelli

Full Text Available Kinesin motor proteins drive intracellular transport by coupling ATP hydrolysis to conformational changes that mediate directed movement along microtubules. Characterizing these distinct conformations and their interconversion mechanism is essential to determining an atomic-level model of kinesin action. Here we report a comprehensive principal component analysis of 114 experimental structures along with the results of conventional and accelerated molecular dynamics simulations that together map the structural dynamics of the kinesin motor domain. All experimental structures were found to reside in one of three distinct conformational clusters (ATP-like, ADP-like and Eg5 inhibitor-bound. These groups differ in the orientation of key functional elements, most notably the microtubule binding α4-α5, loop8 subdomain and α2b-β4-β6-β7 motor domain tip. Group membership was found not to correlate with the nature of the bound nucleotide in a given structure. However, groupings were coincident with distinct neck-linker orientations. Accelerated molecular dynamics simulations of ATP, ADP and nucleotide free Eg5 indicate that all three nucleotide states could sample the major crystallographically observed conformations. Differences in the dynamic coupling of distal sites were also evident. In multiple ATP bound simulations, the neck-linker, loop8 and the α4-α5 subdomain display correlated motions that are absent in ADP bound simulations. Further dissection of these couplings provides evidence for a network of dynamic communication between the active site, microtubule-binding interface and neck-linker via loop7 and loop13. Additional simulations indicate that the mutations G325A and G326A in loop13 reduce the flexibility of these regions and disrupt their couplings. Our combined results indicate that the reported ATP and ADP-like conformations of kinesin are intrinsically accessible regardless of nucleotide state and support a model where neck

Designer TGFβ superfamily ligands with diversified functionality.

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George P Allendorph

Full Text Available Transforming Growth Factor--beta (TGFβ superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs, and Bone Morphogenetic Proteins (BMPs, are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer, to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values.

Large-Scale Analysis Exploring Evolution of Catalytic Machineries and Mechanisms in Enzyme Superfamilies.

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Furnham, Nicholas; Dawson, Natalie L; Rahman, Syed A; Thornton, Janet M; Orengo, Christine A

2016-01-29

Enzymes, as biological catalysts, form the basis of all forms of life. How these proteins have evolved their functions remains a fundamental question in biology. Over 100 years of detailed biochemistry studies, combined with the large volumes of sequence and protein structural data now available, means that we are able to perform large-scale analyses to address this question. Using a range of computational tools and resources, we have compiled information on all experimentally annotated changes in enzyme function within 379 structurally defined protein domain superfamilies, linking the changes observed in functions during evolution to changes in reaction chemistry. Many superfamilies show changes in function at some level, although one function often dominates one superfamily. We use quantitative measures of changes in reaction chemistry to reveal the various types of chemical changes occurring during evolution and to exemplify these by detailed examples. Additionally, we use structural information of the enzymes active site to examine how different superfamilies have changed their catalytic machinery during evolution. Some superfamilies have changed the reactions they perform without changing catalytic machinery. In others, large changes of enzyme function, in terms of both overall chemistry and substrate specificity, have been brought about by significant changes in catalytic machinery. Interestingly, in some superfamilies, relatives perform similar functions but with different catalytic machineries. This analysis highlights characteristics of functional evolution across a wide range of superfamilies, providing insights that will be useful in predicting the function of uncharacterised sequences and the design of new synthetic enzymes. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Tau excess impairs mitosis and kinesin-5 function, leading to aneuploidy and cell death.

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Bougé, Anne-Laure; Parmentier, Marie-Laure

2016-03-01

In neurodegenerative diseases such as Alzheimer's disease (AD), cell cycle defects and associated aneuploidy have been described. However, the importance of these defects in the physiopathology of AD and the underlying mechanistic processes are largely unknown, in particular with respect to the microtubule (MT)-binding protein Tau, which is found in excess in the brain and cerebrospinal fluid of affected individuals. Although it has long been known that Tau is phosphorylated during mitosis to generate a lower affinity for MTs, there is, to our knowledge, no indication that an excess of this protein could affect mitosis. Here, we studied the effect of an excess of human Tau (hTau) protein on cell mitosis in vivo. Using the Drosophila developing wing disc epithelium as a model, we show that an excess of hTau induces a mitotic arrest, with the presence of monopolar spindles. This mitotic defect leads to aneuploidy and apoptotic cell death. We studied the mechanism of action of hTau and found that the MT-binding domain of hTau is responsible for these defects. We also demonstrate that the effects of hTau occur via the inhibition of the function of the kinesin Klp61F, the Drosophila homologue of kinesin-5 (also called Eg5 or KIF11). We finally show that this deleterious effect of hTau is also found in other Drosophila cell types (neuroblasts) and tissues (the developing eye disc), as well as in human HeLa cells. By demonstrating that MT-bound Tau inhibits the Eg5 kinesin and cell mitosis, our work provides a new framework to consider the role of Tau in neurodegenerative diseases. © 2016. Published by The Company of Biologists Ltd.

Initial conformation of kinesin's neck linker

International Nuclear Information System (INIS)

Geng Yi-Zhao; Yan Shi-Wei; Ji Qing; Liu Shu-Xia

2014-01-01

How ATP binding initiates the docking process of kinesin's neck linker is a key question in understanding kinesin mechanisms. By exploiting a molecular dynamics method, we investigate the initial conformation of kinesin's neck linker in its docking process. We find that, in the initial conformation, the neck linker has interactions with β0 and forms a ‘cover-neck bundle’ structure with β0. From this initial structure, the formation of extra turns and the docking of the cover-neck bundle structure can be achieved. The motor head provides a forward force on the initial cover-neck bundle structure through ATP-induced rotation. This force, together with the hydrophobic interaction of ILE327 with the hydrophobic pocket on the motor head, drives the formation of the extra turn and initiates the neck linker docking process. Based on these findings, a pathway from ATP binding-induced motor head rotation to neck linker docking is proposed. (interdisciplinary physics and related areas of science and technology)

Evolution of plant virus movement proteins from the 30K superfamily and of their homologs integrated in plant genomes

Energy Technology Data Exchange (ETDEWEB)

Mushegian, Arcady R., E-mail: mushegian2@gmail.com [Division of Molecular and Cellular Biosciences, National Science Foundation, 4201 Wilson Boulevard, Arlington, VA 22230 (United States); Elena, Santiago F., E-mail: sfelena@ibmcp.upv.es [Instituto de Biología Molecular y Celular de Plantas, CSIC-UPV, 46022 València (Spain); The Santa Fe Institute, Santa Fe, NM 87501 (United States)

2015-02-15

Homologs of Tobacco mosaic virus 30K cell-to-cell movement protein are encoded by diverse plant viruses. Mechanisms of action and evolutionary origins of these proteins remain obscure. We expand the picture of conservation and evolution of the 30K proteins, producing sequence alignment of the 30K superfamily with the broadest phylogenetic coverage thus far and illuminating structural features of the core all-beta fold of these proteins. Integrated copies of pararetrovirus 30K movement genes are prevalent in euphyllophytes, with at least one copy intact in nearly every examined species, and mRNAs detected for most of them. Sequence analysis suggests repeated integrations, pseudogenizations, and positive selection in those provirus genes. An unannotated 30K-superfamily gene in Arabidopsis thaliana genome is likely expressed as a fusion with the At1g37113 transcript. This molecular background of endopararetrovirus gene products in plants may change our view of virus infection and pathogenesis, and perhaps of cellular homeostasis in the hosts. - Highlights: • Sequence region shared by plant virus “30K” movement proteins has an all-beta fold. • Most euphyllophyte genomes contain integrated copies of pararetroviruses. • These integrated virus genomes often include intact movement protein genes. • Molecular evidence suggests that these “30K” genes may be selected for function.

Tracing the Evolutionary History of the CAP Superfamily of Proteins Using Amino Acid Sequence Homology and Conservation of Splice Sites.

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Abraham, Anup; Chandler, Douglas E

2017-10-01

Proteins of the CAP superfamily play numerous roles in reproduction, innate immune responses, cancer biology, and venom toxicology. Here we document the breadth of the CAP (Cysteine-RIch Secretory Protein (CRISP), Antigen 5, and Pathogenesis-Related) protein superfamily and trace the major events in its evolution using amino acid sequence homology and the positions of exon/intron borders within their genes. Seldom acknowledged in the literature, we find that many of the CAP subfamilies present in mammals, where they were originally characterized, have distinct homologues in the invertebrate phyla. Early eukaryotic CAP genes contained only one exon inherited from prokaryotic predecessors and as evolution progressed an increasing number of introns were inserted, reaching 2-5 in the invertebrate world and 5-15 in the vertebrate world. Focusing on the CRISP subfamily, we propose that these proteins evolved in three major steps: (1) origination of the CAP/PR/SCP domain in bacteria, (2) addition of a small Hinge domain to produce the two-domain SCP-like proteins found in roundworms and anthropoids, and (3) addition of an Ion Channel Regulatory domain, borrowed from invertebrate peptide toxins, to produce full length, three-domain CRISP proteins, first seen in insects and later to diversify into multiple subtypes in the vertebrate world.

Bidirectional motility of the fission yeast kinesin-5, Cut7

Energy Technology Data Exchange (ETDEWEB)

Edamatsu, Masaki, E-mail: cedam@mail.ecc.u-tokyo.ac.jp

2014-03-28

Highlights: • Motile properties of Cut7 (fission yeast kinesin-5) were studied for the first time. • Half-length Cut7 moved toward plus-end direction of microtubule. • Full-length Cut7 moved toward minus-end direction of microtubule. • N- and C-terminal microtubule binding sites did not switch the motile direction. - Abstract: Kinesin-5 is a homotetrameric motor with its motor domain at the N-terminus. Kinesin-5 crosslinks microtubules and functions in separating spindle poles during mitosis. In this study, the motile properties of Cut7, fission yeast kinesin-5, were examined for the first time. In in vitro motility assays, full-length Cut7 moved toward minus-end of microtubules, but the N-terminal half of Cut7 moved toward the opposite direction. Furthermore, additional truncated constructs lacking the N-terminal or C-terminal regions, but still contained the motor domain, did not switch the motile direction. These indicated that Cut7 was a bidirectional motor, and microtubule binding regions at the N-terminus and C-terminus were not involved in its directionality.

Delivery of kinesin spindle protein targeting siRNA in solid lipid nanoparticles to cellular models of tumor vasculature

International Nuclear Information System (INIS)

Ying, Bo; Campbell, Robert B.

2014-01-01

Highlights: • siRNA-lipid nanoparticles are solid particles not lipid bilayers with aqueous core. • High, but not low, PEG content can prevent nanoparticle encapsulation of siRNA. • PEG reduces cellular toxicity of cationic nanoparticles in vitro. • PEG reduces zeta potential while improving gene silencing of siRNA nanoparticles. • Kinesin spindle protein can be an effective target for tumor vascular targeting. - Abstract: The ideal siRNA delivery system should selectively deliver the construct to the target cell, avoid enzymatic degradation, and evade uptake by phagocytes. In the present study, we evaluated the importance of polyethylene glycol (PEG) on lipid-based carrier systems for encapsulating, and delivering, siRNA to tumor vessels using cellular models. Lipid nanoparticles containing different percentage of PEG were evaluated based on their physical chemical properties, density compared to water, siRNA encapsulation, toxicity, targeting efficiency and gene silencing in vitro. siRNA can be efficiently loaded into lipid nanoparticles (LNPs) when DOTAP is included in the formulation mixture. However, the total amount encapsulated decreased with increase in PEG content. In the presence of siRNA, the final formulations contained a mixed population of particles based on density. The major population which contains the majority of siRNA exhibited a density of 4% glucose, and the minor fraction associated with a decreased amount of siRNA had a density less than PBS. The inclusion of 10 mol% PEG resulted in a greater amount of siRNA associated with the minor fraction. Finally, when kinesin spindle protein (KSP) siRNA was encapsulated in lipid nanoparticles containing a modest amount of PEG, the proliferation of endothelial cells was inhibited due to the efficient knock down of KSP mRNA. The presence of siRNA resulted in the formation of solid lipid nanoparticles when prepared using the thin film and hydration method. LNPs with a relatively modest amount of

Structure-based molecular simulations reveal the enhancement of biased Brownian motions in single-headed kinesin.

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Kanada, Ryo; Kuwata, Takeshi; Kenzaki, Hiroo; Takada, Shoji

2013-01-01

Kinesin is a family of molecular motors that move unidirectionally along microtubules (MT) using ATP hydrolysis free energy. In the family, the conventional two-headed kinesin was experimentally characterized to move unidirectionally through "walking" in a hand-over-hand fashion by coordinated motions of the two heads. Interestingly a single-headed kinesin, a truncated KIF1A, still can generate a biased Brownian movement along MT, as observed by in vitro single molecule experiments. Thus, KIF1A must use a different mechanism from the conventional kinesin to achieve the unidirectional motions. Based on the energy landscape view of proteins, for the first time, we conducted a set of molecular simulations of the truncated KIF1A movements over an ATP hydrolysis cycle and found a mechanism exhibiting and enhancing stochastic forward-biased movements in a similar way to those in experiments. First, simulating stand-alone KIF1A, we did not find any biased movements, while we found that KIF1A with a large friction cargo-analog attached to the C-terminus can generate clearly biased Brownian movements upon an ATP hydrolysis cycle. The linked cargo-analog enhanced the detachment of the KIF1A from MT. Once detached, diffusion of the KIF1A head was restricted around the large cargo which was located in front of the head at the time of detachment, thus generating a forward bias of the diffusion. The cargo plays the role of a diffusional anchor, or cane, in KIF1A "walking."

Structure-based molecular simulations reveal the enhancement of biased Brownian motions in single-headed kinesin.

Directory of Open Access Journals (Sweden)

Ryo Kanada

Full Text Available Kinesin is a family of molecular motors that move unidirectionally along microtubules (MT using ATP hydrolysis free energy. In the family, the conventional two-headed kinesin was experimentally characterized to move unidirectionally through "walking" in a hand-over-hand fashion by coordinated motions of the two heads. Interestingly a single-headed kinesin, a truncated KIF1A, still can generate a biased Brownian movement along MT, as observed by in vitro single molecule experiments. Thus, KIF1A must use a different mechanism from the conventional kinesin to achieve the unidirectional motions. Based on the energy landscape view of proteins, for the first time, we conducted a set of molecular simulations of the truncated KIF1A movements over an ATP hydrolysis cycle and found a mechanism exhibiting and enhancing stochastic forward-biased movements in a similar way to those in experiments. First, simulating stand-alone KIF1A, we did not find any biased movements, while we found that KIF1A with a large friction cargo-analog attached to the C-terminus can generate clearly biased Brownian movements upon an ATP hydrolysis cycle. The linked cargo-analog enhanced the detachment of the KIF1A from MT. Once detached, diffusion of the KIF1A head was restricted around the large cargo which was located in front of the head at the time of detachment, thus generating a forward bias of the diffusion. The cargo plays the role of a diffusional anchor, or cane, in KIF1A "walking."

Functional diversity of the superfamily of K⁺ transporters to meet various requirements.

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Diskowski, Marina; Mikusevic, Vedrana; Stock, Charlott; Hänelt, Inga

2015-09-01

The superfamily of K+ transporters unites proteins from plants, fungi, bacteria, and archaea that translocate K+ and/or Na+ across membranes. These proteins are key components in osmotic regulation, pH homeostasis, and resistance to high salinity and dryness. The members of the superfamily are closely related to K+ channels such as KcsA but also show several striking differences that are attributed to their altered functions. This review highlights these functional differences, focusing on the bacterial superfamily members KtrB, TrkH, and KdpA. The functional variations within the family and comparison to MPM-type K+ channels are discussed in light of the recently solved structures of the Ktr and Trk systems.

Evolutionary history, structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes.

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Anantharaman, Vivek; Aravind, L

2003-01-01

Peptidoglycan is hydrolyzed by a diverse set of enzymes during bacterial growth, development and cell division. The N1pC/P60 proteins define a family of cell-wall peptidases that are widely represented in various bacterial lineages. Currently characterized members are known to hydrolyze D-gamma-glutamyl-meso-diaminopimelate or N-acetylmuramate-L-alanine linkages. Detailed analysis of the N1pC/P60 peptidases showed that these proteins define a large superfamily encompassing several diverse groups of proteins. In addition to the well characterized P60-like proteins, this superfamily includes the AcmB/LytN and YaeF/YiiX families of bacterial proteins, the amidase domain of bacterial and kinetoplastid glutathionylspermidine synthases (GSPSs), and several proteins from eukaryotes, phages, poxviruses, positive-strand RNA viruses, and certain archaea. The eukaryotic members include lecithin retinol acyltransferase (LRAT), nematode developmental regulator Egl-26, and candidate tumor suppressor H-rev107. These eukaryotic proteins, along with the bacterial YaeF/poxviral G6R family, show a circular permutation of the catalytic domain. We identified three conserved residues, namely a cysteine, a histidine and a polar residue, that are involved in the catalytic activities of this superfamily. Evolutionary analysis of this superfamily shows that it comprises four major families, with diverse domain architectures in each of them. Several related, but distinct, catalytic activities, such as murein degradation, acyl transfer and amide hydrolysis, have emerged in the N1pC/P60 superfamily. The three conserved catalytic residues of this superfamily are shown to be equivalent to the catalytic triad of the papain-like thiol peptidases. The predicted structural features indicate that the N1pC/P60 enzymes contain a fold similar to the papain-like peptidases, transglutaminases and arylamine acetyltransferases.

The kinesin AtPSS1 promotes synapsis and is required for proper crossover distribution in meiosis.

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Yann Duroc

2014-10-01

Full Text Available Meiotic crossovers (COs shape genetic diversity by mixing homologous chromosomes at each generation. CO distribution is a highly regulated process. CO assurance forces the occurrence of at least one obligatory CO per chromosome pair, CO homeostasis smoothes out the number of COs when faced with variation in precursor number and CO interference keeps multiple COs away from each other along a chromosome. In several organisms, it has been shown that cytoskeleton forces are transduced to the meiotic nucleus via KASH- and SUN-domain proteins, to promote chromosome synapsis and recombination. Here we show that the Arabidopsis kinesin AtPSS1 plays a major role in chromosome synapsis and regulation of CO distribution. In Atpss1 meiotic cells, chromosome axes and DNA double strand breaks (DSBs appear to form normally but only a variable portion of the genome synapses and is competent for CO formation. Some chromosomes fail to form the obligatory CO, while there is an increased CO density in competent regions. However, the total number of COs per cell is unaffected. We further show that the kinesin motor domain of AtPSS1 is required for its meiotic function, and that AtPSS1 interacts directly with WIP1 and WIP2, two KASH-domain proteins. Finally, meiocytes missing AtPSS1 and/or SUN proteins show similar meiotic defects suggesting that AtPSS1 and SUNs act in the same pathway. This suggests that forces produced by the AtPSS1 kinesin and transduced by WIPs/SUNs, are required to authorize complete synapsis and regulate maturation of recombination intermediates into COs. We suggest that a form of homeostasis applies, which maintains the total number of COs per cell even if only a part of the genome is competent for CO formation.

Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

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Lu, Wen; Winding, Michael; Lakonishok, Margot; Wildonger, Jill

2016-01-01

Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants. PMID:27512034

Oligomerisation status and evolutionary conservation of interfaces of protein structural domain superfamilies.

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Sukhwal, Anshul; Sowdhamini, Ramanathan

2013-07-01

and its remote homologue-interacting partner pair. We found that, in exceptional cases, homologous proteins belonging to the same superfamily, but with remote sequence similarity, can share similar interfaces.

MP20, the second most abundant lens membrane protein and member of the tetraspanin superfamily, joins the list of ligands of galectin-3

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Donaldson Paul J

2001-08-01

Full Text Available Abstract Background Although MP20 is the second most highly expressed membrane protein in the lens its function remains an enigma. Putative functions for MP20 have recently been inferred from its assignment to the tetraspanin superfamily of integral membrane proteins. Members of this family have been shown to be involved in cellular proliferation, differentiation, migration, and adhesion. In this study, we show that MP20 associates with galectin-3, a known adhesion modulator. Results MP20 and galectin-3 co-localized in selected areas of the lens fiber cell plasma membrane. Individually, these proteins purified with apparent molecular masses of 60 kDa and 22 kDa, respectively. A 104 kDa complex was formed in vitro upon mixing the purified proteins. A 102 kDa complex of MP20 and galectin-3 could also be isolated from detergent-solubilized native fiber cell membranes. Binding between MP20 and galectin-3 was disrupted by lactose suggesting the lectin site was involved in the interaction. Conclusions MP20 adds to a growing list of ligands of galectin-3 and appears to be the first representative of the tetraspanin superfamily identified to possess this specificity.

Lemur tyrosine kinase-2 signalling regulates kinesin-1 light chain-2 phosphorylation and binding of Smad2 cargo.

LENUS (Irish Health Repository)

Manser, C

2012-05-31

A recent genome-wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9, but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3β (GSK3β). KLC2 phosphorylation by GSK3β induces the release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3β on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-β (TGFβ) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFβ-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling.

Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily

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Matsunaga, James; Barocchi, Michele A.; Croda, Julio; Young, Tracy A.; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A.; Reis, Mitermayer G.; Riley, Lee W.; Haake, David A.; Ko, Albert I.

2005-01-01

Summary Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudo-gene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis. PMID:12890019

BORC Functions Upstream of Kinesins 1 and 3 to Coordinate Regional Movement of Lysosomes along Different Microtubule Tracks.

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Guardia, Carlos M; Farías, Ginny G; Jia, Rui; Pu, Jing; Bonifacino, Juan S

2016-11-15

The multiple functions of lysosomes are critically dependent on their ability to undergo bidirectional movement along microtubules between the center and the periphery of the cell. Centrifugal and centripetal movement of lysosomes is mediated by kinesin and dynein motors, respectively. We recently described a multi-subunit complex named BORC that recruits the small GTPase Arl8 to lysosomes to promote their kinesin-dependent movement toward the cell periphery. Here, we show that BORC and Arl8 function upstream of two structurally distinct kinesin types: kinesin-1 (KIF5B) and kinesin-3 (KIF1Bβ and KIF1A). Remarkably, KIF5B preferentially moves lysosomes on perinuclear tracks enriched in acetylated α-tubulin, whereas KIF1Bβ and KIF1A drive lysosome movement on more rectilinear, peripheral tracks enriched in tyrosinated α-tubulin. These findings establish BORC as a master regulator of lysosome positioning through coupling to different kinesins and microtubule tracks. Common regulation by BORC enables coordinate control of lysosome movement in different regions of the cell. Published by Elsevier Inc.

Monte Carlo analysis of neck linker extension in kinesin molecular motors.

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Matthew L Kutys

2010-11-01

Full Text Available Kinesin stepping is thought to involve both concerted conformational changes and diffusive movement, but the relative roles played by these two processes are not clear. The neck linker docking model is widely accepted in the field, but the remainder of the step--diffusion of the tethered head to the next binding site--is often assumed to occur rapidly with little mechanical resistance. Here, we investigate the effect of tethering by the neck linker on the diffusive movement of the kinesin head, and focus on the predicted behavior of motors with naturally or artificially extended neck linker domains. The kinesin chemomechanical cycle was modeled using a discrete-state Markov chain to describe chemical transitions. Brownian dynamics were used to model the tethered diffusion of the free head, incorporating resistive forces from the neck linker and a position-dependent microtubule binding rate. The Brownian dynamics and chemomechanical cycle were coupled to model processive runs consisting of many 8 nm steps. Three mechanical models of the neck linker were investigated: Constant Stiffness (a simple spring, Increasing Stiffness (analogous to a Worm-Like Chain, and Reflecting (negligible stiffness up to a limiting contour length. Motor velocities and run lengths from simulated paths were compared to experimental results from Kinesin-1 and a mutant containing an extended neck linker domain. When tethered by an increasingly stiff spring, the head is predicted to spend an unrealistically short amount of time within the binding zone, and extending the neck is predicted to increase both the velocity and processivity, contrary to experiments. These results suggest that the Worm-Like Chain is not an adequate model for the flexible neck linker domain. The model can be reconciled with experimental data if the neck linker is either much more compliant or much stiffer than generally assumed, or if weak kinesin-microtubule interactions stabilize the diffusing

Dynamic microtubule organization and mitochondrial transport are regulated by distinct Kinesin-1 pathways

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Anna Melkov

2015-12-01

Full Text Available The microtubule (MT plus-end motor kinesin heavy chain (Khc is well known for its role in long distance cargo transport. Recent evidence showed that Khc is also required for the organization of the cellular MT network by mediating MT sliding. We found that mutations in Khc and the gene of its adaptor protein, kinesin light chain (Klc resulted in identical bristle morphology defects, with the upper part of the bristle being thinner and flatter than normal and failing to taper towards the bristle tip. We demonstrate that bristle mitochondria transport requires Khc but not Klc as a competing force to dynein heavy chain (Dhc. Surprisingly, we demonstrate for the first time that Dhc is the primary motor for both anterograde and retrograde fast mitochondria transport. We found that the upper part of Khc and Klc mutant bristles lacked stable MTs. When following dynamic MT polymerization via the use of GFP-tagged end-binding protein 1 (EB1, it was noted that at Khc and Klc mutant bristle tips, dynamic MTs significantly deviated from the bristle parallel growth axis, relative to wild-type bristles. We also observed that GFP-EB1 failed to concentrate as a focus at the tip of Khc and Klc mutant bristles. We propose that the failure of bristle tapering is due to defects in directing dynamic MTs at the growing tip. Thus, we reveal a new function for Khc and Klc in directing dynamic MTs during polarized cell growth. Moreover, we also demonstrate a novel mode of coordination in mitochondrial transport between Khc and Dhc.

A hereditary spastic paraplegia mutation in kinesin-1A/KIF5A disrupts neurofilament transport

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Brown Anthony

2010-11-01

Full Text Available Abstract Background Hereditary spastic paraplegias are a group of neurological disorders characterized by progressive distal degeneration of the longest ascending and descending axons in the spinal cord, leading to lower limb spasticity and weakness. One of the dominantly inherited forms of this disease (spastic gait type 10, or SPG10 is caused by point mutations in kinesin-1A (also known as KIF5A, which is thought to be an anterograde motor for neurofilaments. Results We investigated the effect of an SPG10 mutation in kinesin-1A (N256S-kinesin-1A on neurofilament transport in cultured mouse cortical neurons using live-cell fluorescent imaging. N256S-kinesin-1A decreased both anterograde and retrograde neurofilament transport flux by decreasing the frequency of anterograde and retrograde movements. Anterograde velocity was not affected, whereas retrograde velocity actually increased. Conclusions These data reveal subtle complexities to the functional interdependence of the anterograde and retrograde neurofilament motors and they also raise the possibility that anterograde and retrograde neurofilament transport may be disrupted in patients with SPG10.

Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array

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Schuster, M.; Kilaru, S.; Fink, G.; Collemare, J.A.R.; Roger, Y.; Steinberg, G.

2011-01-01

The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to

Ribosomal protein NtRPL17 interacts with kinesin-12 family protein NtKRP and functions in the regulation of embryo/seed size and radicle growth.

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Tian, Shujuan; Wu, Jingjing; Liu, Yuan; Huang, Xiaorong; Li, Fen; Wang, Zhaodan; Sun, Meng-Xiang

2017-11-28

We previously reported that a novel motor protein belonging to the kinesin-12 family, NtKRP, displays critical roles in regulating embryo and seed size establishment. However, it remains unknown exactly how NtKRP contributes to this developmental process. Here, we report that a 60S ribosomal protein NtRPL17 directly interacts with NtKRP. The phenotypes of NtRPL17 RNAi lines show notable embryo and seed size reduction. Structural observations of the NtRPL17-silenced embryos/seeds reveal that the embryo size reduction is due to a decrease in cell number. In these embryos, cell division cycle progression is delayed at the G2/M transition. These phenotypes are similar to that in NtKRP-silenced embryos/seeds, indicating that NtKRP and NtRPL17 function as partners in the same regulatory pathway during seed development and specifically regulate cell cycle progression to control embryo/seed size. This work reveals that NtRPL17, as a widely distributed ribosomal protein, plays a critical role in seed development and provides a new clue in the regulation of seed size. Confirmation of the interaction between NtKRP and NtRPL17 and their co-function in the control of the cell cycle also suggests that the mechanism might be conserved in both plants and animals. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Phosphatidylcholine transfer protein interacts with thioesterase superfamily member 2 to attenuate insulin signaling.

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Ersoy, Baran A; Tarun, Akansha; D'Aquino, Katharine; Hancer, Nancy J; Ukomadu, Chinweike; White, Morris F; Michel, Thomas; Manning, Brendan D; Cohen, David E

2013-07-30

Phosphatidylcholine transfer protein (PC-TP) is a phospholipid-binding protein that is enriched in liver and that interacts with thioesterase superfamily member 2 (THEM2). Mice lacking either protein exhibit improved hepatic glucose homeostasis and are resistant to diet-induced diabetes. Insulin receptor substrate 2 (IRS2) and mammalian target of rapamycin complex 1 (mTORC1) are key effectors of insulin signaling, which is attenuated in diabetes. We found that PC-TP inhibited IRS2, as evidenced by insulin-independent IRS2 activation after knockdown, genetic ablation, or chemical inhibition of PC-TP. In addition, IRS2 was activated after knockdown of THEM2, providing support for a role for the interaction of PC-TP with THEM2 in suppressing insulin signaling. Additionally, we showed that PC-TP bound to tuberous sclerosis complex 2 (TSC2) and stabilized the components of the TSC1-TSC2 complex, which functions to inhibit mTORC1. Preventing phosphatidylcholine from binding to PC-TP disrupted interactions of PC-TP with THEM2 and TSC2, and disruption of the PC-TP-THEM2 complex was associated with increased activation of both IRS2 and mTORC1. In livers of mice with genetic ablation of PC-TP or that had been treated with a PC-TP inhibitor, steady-state amounts of IRS2 were increased, whereas those of TSC2 were decreased. These findings reveal a phospholipid-dependent mechanism that suppresses insulin signaling downstream of its receptor.

Hypothesis: NDL proteins function in stress responses by regulating microtubule organization.

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Khatri, Nisha; Mudgil, Yashwanti

2015-01-01

N-MYC DOWNREGULATED-LIKE proteins (NDL), members of the alpha/beta hydrolase superfamily were recently rediscovered as interactors of G-protein signaling in Arabidopsis thaliana. Although the precise molecular function of NDL proteins is still elusive, in animals these proteins play protective role in hypoxia and expression is induced by hypoxia and nickel, indicating role in stress. Homology of NDL1 with animal counterpart N-MYC DOWNREGULATED GENE (NDRG) suggests similar functions in animals and plants. It is well established that stress responses leads to the microtubule depolymerization and reorganization which is crucial for stress tolerance. NDRG is a microtubule-associated protein which mediates the microtubule organization in animals by causing acetylation and increases the stability of α-tubulin. As NDL1 is highly homologous to NDRG, involvement of NDL1 in the microtubule organization during plant stress can also be expected. Discovery of interaction of NDL with protein kinesin light chain- related 1, enodomembrane family protein 70, syntaxin-23, tubulin alpha-2 chain, as a part of G protein interactome initiative encourages us to postulate microtubule stabilizing functions for NDL family in plants. Our search for NDL interactors in G protein interactome also predicts the role of NDL proteins in abiotic stress tolerance management. Based on published report in animals and predicted interacting partners for NDL in G protein interactome lead us to hypothesize involvement of NDL in the microtubule organization during abiotic stress management in plants.

TNF and TNF Receptor Superfamily Members in HIV infection: New Cellular Targets for Therapy?

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Amit Kumar

2013-01-01

Full Text Available Tumor necrosis factor (TNF and TNF receptors (TNFR superfamily members are engaged in diverse cellular phenomena such as cellular proliferation, morphogenesis, apoptosis, inflammation, and immune regulation. Their role in regulating viral infections has been well documented. Viruses have evolved with numerous strategies to interfere with TNF-mediated signaling indicating the importance of TNF and TNFR superfamily in viral pathogenesis. Recent research reports suggest that TNF and TNFRs play an important role in the pathogenesis of HIV. TNFR signaling modulates HIV replication and HIV proteins interfere with TNF/TNFR pathways. Since immune activation and inflammation are the hallmark of HIV infection, the use of TNF inhibitors can have significant impact on HIV disease progression. In this review, we will describe how HIV infection is modulated by signaling mediated through members of TNF and TNFR superfamily and in turn how these latter could be targeted by HIV proteins. Finally, we will discuss the emerging therapeutics options based on modulation of TNF activity that could ultimately lead to the cure of HIV-infected patients.

Isolation of a novel LPS-induced component of the ML superfamily in Ciona intestinalis.

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Vizzini, Aiti; Bonura, Angela; Longo, Valeria; Sanfratello, Maria Antonietta; Parrinello, Daniela; Cammarata, Matteo; Colombo, Paolo

2015-11-01

ML superfamily represents a group of proteins playing important roles in lipid metabolism and innate immune response. In this study, we report the identification of the first component of the ML superfamily in the invertebrate Ciona intestinalis by means of a subtractive hybridization strategy. Sequence homology and phylogenetic analysis showed that this protein forms a specific clade with vertebrate components of the Niemann-Pick type C2 protein and, for this reason, it has been named Ci-NPC2. The putative Ci-NPC2 is a 150 amino acids long protein with a short signal peptide, seven cysteine residues, three putative lipid binding site and a three-dimensional model showing a characteristic β-strand structure. Gene expression analysis demonstrated that the Ci-NPC2 protein is positively upregulated after LPS inoculum with a peak of expression 1 h after challenge. Finally, in-situ hybridization demonstrated that the Ci-NPC2 protein is preferentially expressed in hemocytes inside the vessel lumen. Copyright © 2015 Elsevier Ltd. All rights reserved.

Detectable states, cycle fluxes, and motility scaling of molecular motor kinesin: An integrative kinetic graph theory analysis

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Ren, Jie

2017-12-01

The process by which a kinesin motor couples its ATPase activity with concerted mechanical hand-over-hand steps is a foremost topic of molecular motor physics. Two major routes toward elucidating kinesin mechanisms are the motility performance characterization of velocity and run length, and single-molecular state detection experiments. However, these two sets of experimental approaches are largely uncoupled to date. Here, we introduce an integrative motility state analysis based on a theorized kinetic graph theory for kinesin, which, on one hand, is validated by a wealth of accumulated motility data, and, on the other hand, allows for rigorous quantification of state occurrences and chemomechanical cycling probabilities. An interesting linear scaling for kinesin motility performance across species is discussed as well. An integrative kinetic graph theory analysis provides a powerful tool to bridge motility and state characterization experiments, so as to forge a unified effort for the elucidation of the working mechanisms of molecular motors.

Deletion of the Tail Domain of the Kinesin-5 Cin8 Affects Its Directionality*

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Düselder, André; Fridman, Vladimir; Thiede, Christina; Wiesbaum, Alice; Goldstein, Alina; Klopfenstein, Dieter R.; Zaitseva, Olga; Janson, Marcel E.; Gheber, Larisa; Schmidt, Christoph F.

2015-01-01

The bipolar kinesin-5 motors are one of the major players that govern mitotic spindle dynamics. Their bipolar structure enables them to cross-link and slide apart antiparallel microtubules (MTs) emanating from the opposing spindle poles. The budding yeast kinesin-5 Cin8 was shown to switch from fast minus-end- to slow plus-end-directed motility upon binding between antiparallel MTs. This unexpected finding revealed a new dimension of cellular control of transport, the mechanism of which is unknown. Here we have examined the role of the C-terminal tail domain of Cin8 in regulating directionality. We first constructed a stable dimeric Cin8/kinesin-1 chimera (Cin8Kin), consisting of head and neck linker of Cin8 fused to the stalk of kinesin-1. As a single dimeric motor, Cin8Kin switched frequently between plus and minus directionality along single MTs, demonstrating that the Cin8 head domains are inherently bidirectional, but control over directionality was lost. We next examined the activity of a tetrameric Cin8 lacking only the tail domains (Cin8Δtail). In contrast to wild-type Cin8, the motility of single molecules of Cin8Δtail in high ionic strength was slow and bidirectional, with almost no directionality switches. Cin8Δtail showed only a weak ability to cross-link MTs in vitro. In vivo, Cin8Δtail exhibited bias toward the plus-end of the MTs and was unable to support viability of cells as the sole kinesin-5 motor. We conclude that the tail of Cin8 is not necessary for bidirectional processive motion, but is controlling the switch between plus- and minus-end-directed motility. PMID:25991727

Crystal Structures of the Tetratricopeptide Repeat Domains of Kinesin Light Chains: Insight into Cargo Recognition Mechanisms

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Zhu, Haizhong; Lee, Han Youl; Tong, Yufeng; Hong, Bum-Soo; Kim, Kyung-Phil; Shen, Yang; Lim, Kyung Jik; Mackenzie, Farrell; Tempel, Wolfram; Park, Hee-Won (SGC-Toronto); (PPCS); (Toronto)

2012-10-23

Kinesin-1 transports various cargos along the axon by interacting with the cargos through its light chain subunit. Kinesin light chains (KLC) utilize its tetratricopeptide repeat (TPR) domain to interact with over 10 different cargos. Despite a high sequence identity between their TPR domains (87%), KLC1 and KLC2 isoforms exhibit differential binding properties towards some cargos. We determined the structures of human KLC1 and KLC2 tetratricopeptide repeat (TPR) domains using X-ray crystallography and investigated the different mechanisms by which KLCs interact with their cargos. Using isothermal titration calorimetry, we attributed the specific interaction between KLC1 and JNK-interacting protein 1 (JIP1) cargo to residue N343 in the fourth TRP repeat. Structurally, the N343 residue is adjacent to other asparagines and lysines, creating a positively charged polar patch within the groove of the TPR domain. Whereas, KLC2 with the corresponding residue S328 did not interact with JIP1. Based on these finding, we propose that N343 of KLC1 can form 'a carboxylate clamp' with its neighboring asparagine to interact with JIP1, similar to that of HSP70/HSP90 organizing protein-1's (HOP1) interaction with heat shock proteins. For the binding of cargos shared by KLC1 and KLC2, we propose a different site located within the groove but not involving N343. We further propose a third binding site on KLC1 which involves a stretch of polar residues along the inter-TPR loops that may form a network of hydrogen bonds to JIP3 and JIP4. Together, these results provide structural insights into possible mechanisms of interaction between KLC TPR domains and various cargo proteins.

Structural plasticity of the N-terminal capping helix of the TPR domain of kinesin light chain.

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The Quyen Nguyen

Full Text Available Kinesin1 plays a major role in neuronal transport by recruiting many different cargos through its kinesin light chain (KLC. Various structurally unrelated cargos interact with the conserved tetratricopeptide repeat (TPR domain of KLC. The N-terminal capping helix of the TPR domain exhibits an atypical sequence and structural features that may contribute to the versatility of the TPR domain to bind different cargos. We determined crystal structures of the TPR domain of both KLC1 and KLC2 encompassing the N-terminal capping helix and show that this helix exhibits two distinct and defined orientations relative to the rest of the TPR domain. Such a difference in orientation gives rise, at the N-terminal part of the groove, to the formation of one hydrophobic pocket, as well as to electrostatic variations at the groove surface. We present a comprehensive structural analysis of available KLC1/2-TPR domain structures that highlights that ligand binding into the groove can be specific of one or the other N-terminal capping helix orientations. Further, structural analysis reveals that the N-terminal capping helix is always involved in crystal packing contacts, especially in a TPR1:TPR1' contact which highlights its propensity to be a protein-protein interaction site. Together, these results underline that the structural plasticity of the N-terminal capping helix might represent a structural determinant for TPR domain structural versatility in cargo binding.

Ensembler: Enabling High-Throughput Molecular Simulations at the Superfamily Scale.

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Daniel L Parton

2016-06-01

Full Text Available The rapidly expanding body of available genomic and protein structural data provides a rich resource for understanding protein dynamics with biomolecular simulation. While computational infrastructure has grown rapidly, simulations on an omics scale are not yet widespread, primarily because software infrastructure to enable simulations at this scale has not kept pace. It should now be possible to study protein dynamics across entire (superfamilies, exploiting both available structural biology data and conformational similarities across homologous proteins. Here, we present a new tool for enabling high-throughput simulation in the genomics era. Ensembler takes any set of sequences-from a single sequence to an entire superfamily-and shepherds them through various stages of modeling and refinement to produce simulation-ready structures. This includes comparative modeling to all relevant PDB structures (which may span multiple conformational states of interest, reconstruction of missing loops, addition of missing atoms, culling of nearly identical structures, assignment of appropriate protonation states, solvation in explicit solvent, and refinement and filtering with molecular simulation to ensure stable simulation. The output of this pipeline is an ensemble of structures ready for subsequent molecular simulations using computer clusters, supercomputers, or distributed computing projects like Folding@home. Ensembler thus automates much of the time-consuming process of preparing protein models suitable for simulation, while allowing scalability up to entire superfamilies. A particular advantage of this approach can be found in the construction of kinetic models of conformational dynamics-such as Markov state models (MSMs-which benefit from a diverse array of initial configurations that span the accessible conformational states to aid sampling. We demonstrate the power of this approach by constructing models for all catalytic domains in the human

The ATPase of the phi29 DNA packaging motor is a member of the hexameric AAA+ superfamily.

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Schwartz, Chad; De Donatis, Gian Marco; Fang, Huaming; Guo, Peixuan

2013-08-15

The AAA+ superfamily of proteins is a class of motor ATPases performing a wide range of functions that typically exist as hexamers. The ATPase of phi29 DNA packaging motor has long been a subject of debate in terms of stoichiometry and mechanism of action. Here, we confirmed the stoichiometry of phi29 motor ATPase to be a hexamer and provide data suggesting that the phi29 motor ATPase is a member of the classical hexameric AAA+ superfamily. Native PAGE, EMSA, capillary electrophoresis, ATP titration, and binomial distribution assay show that the ATPase is a hexamer. Mutations in the known Walker motifs of the ATPase validated our previous assumptions that the protein exists as another member of this AAA+ superfamily. Our data also supports the finding that the phi29 DNA packaging motor uses a revolution mechanism without rotation or coiling (Schwartz et al., this issue). Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

In silico identification, phylogeny and expression analysis of expansin superfamily in Medicago truncatula

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Ying Liu

2016-01-01

Full Text Available Expansins are important components of plant cell walls, which are involved in the process of cell wall loosening under low extracellular pH. By using a combinational method for homology search and protein domain analysis, a total of 42 expansin genes were identified from Medicago truncatula genome in this study. They were divided into four families, based on sequence alignment and phylogenetic analysis. Gene duplication events were identified in the expansins superfamily, especially in the extension of α-expansin family. By analysis of RNA-sequencing data from National Center for Biotechnology Information, the expansin (EXP genes expressed during tissues development were characterized. Meanwhile, lots of cis-acting regulatory DNA elements in the EXP superfamily were identified, which were mainly related to plant growth and development processes. The results presented in this study are expected to facilitate further research works on this gene superfamily and provide new insights about the molecular mechanisms of expansins in M. truncatula.

Kinesin-73 is a processive motor that localizes to Rab5-containing organelles.

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Huckaba, Thomas M; Gennerich, Arne; Wilhelm, James E; Chishti, Athar H; Vale, Ronald D

2011-03-04

Drosophila Kinesin-73 (Khc-73), which plays a role in mitotic spindle polarity in neuroblasts, is a metazoan-specific member of the Kinesin-3 family of motors, which includes mammalian KIF1A and Caenorhabditis elegans Unc-104. The mechanism of Kinesin-3 motors has been controversial because some studies have reported that they transport cargo as monomers whereas other studies have suggested a dimer mechanism. Here, we have performed single-molecule motility and cell biological studies of Khc-73. We find that constructs containing the motor and the conserved short stretches of putative coiled-coil-forming regions are predominantly monomeric in vitro, but that dimerization allows for fast, processive movement and high force production (7 piconewtons). In Drosophila cell lines, we present evidence that Khc-73 can dimerize in vivo. We also show that Khc-73 is recruited specifically to Rab5-containing endosomes through its "tail" domain. Our results suggest that the N-terminal half of Khc-73 can undergo a monomer-dimer transition to produce a fast processive motor and that its C-terminal half possesses a specific Rab5-vesicle binding domain.

The KAC family of kinesin-like proteins is essential for the association of chloroplasts with the plasma membrane in land plants.

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Suetsugu, Noriyuki; Sato, Yoshikatsu; Tsuboi, Hidenori; Kasahara, Masahiro; Imaizumi, Takato; Kagawa, Takatoshi; Hiwatashi, Yuji; Hasebe, Mitsuyasu; Wada, Masamitsu

2012-11-01

Chloroplasts require association with the plasma membrane for movement in response to light and for appropriate positioning within the cell to capture photosynthetic light efficiently. In Arabidopsis, CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for both the proper movement of chloroplasts and the association of chloroplasts with the plasma membrane, through the reorganization of short actin filaments located on the periphery of the chloroplasts. Here, we show that KAC and CHUP1 orthologs (AcKAC1, AcCHUP1A and AcCHUP1B, and PpKAC1 and PpKAC2) play important roles in chloroplast positioning in the fern Adiantum capillus-veneris and the moss Physcomitrella patens. The knockdown of AcKAC1 and two AcCHUP1 genes induced the aggregation of chloroplasts around the nucleus. Analyses of A. capillus-veneris mutants containing perinuclear-aggregated chloroplasts confirmed that AcKAC1 is required for chloroplast-plasma membrane association. In addition, P. patens lines in which two KAC genes had been knocked out showed an aggregated chloroplast phenotype similar to that of the fern kac1 mutants. These results indicate that chloroplast positioning and movement are mediated through the activities of KAC and CHUP1 proteins, which are conserved in land plants.

An orphan viral TNF receptor superfamily member identified in lymphocystis disease virus.

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Pontejo, Sergio M; Sánchez, Carolina; Martín, Rocío; Mulero, Victoriano; Alcami, Antonio; Alejo, Alí

2013-06-07

Lymphocystis disease virus (LCDV) is a large icosahedral dsDNA-containing virus of the Lymphocystivirus genus within the Iridoviridae family that can cause disease in more than 140 marine and freshwater fish species. While several isolates have been charcaterized and classified into distinct genotypes the complete genomic sequence is currently only available from two species, the LCDV-1, isolated from flounder (Platichtys flesus) in Europe and the LCDV-C, isolated from Japanese cultured flounder (Paralichthys olivaceus) in China. Analysis of the genome of LCDV-C showed it to encode a protein named LDVICp016 with similarities to the Tumour necrosis factor receptor (TNFR) superfamily with immunomodulatory potential. We have expressed and purified the recombinant protein LDVICp016 and screened for potential interaction partners using surface plasmon resonance. Commercially available human and mouse members of the TNF superfamily (TNFSF), along with a representative set of fish-derived TNFSF were tested.We have found the LDVICp016 protein to be secreted and we have identified a second viral TNFR encoded by ORF 095 of the same virus. None of the 42 tested proteins were found to interact with LDVICp016. We show that LDVICp016 is a secreted protein belonging to the TNF receptor family that may be part of a larger gene family in Lymphocystiviruses. While the ligand of this protein remains unknown, possibly due to the species specific nature of this interaction, further investigations into the potential role of this protein in the blockade of immune responses in its fish host are required.

Molecular evolution of the insect chemoreceptor gene superfamily in Drosophila melanogaster

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Robertson, Hugh M.; Warr, Coral G.; Carlson, John R.

2003-01-01

The insect chemoreceptor superfamily in Drosophila melanogaster is predicted to consist of 62 odorant receptor (Or) and 68 gustatory receptor (Gr) proteins, encoded by families of 60 Or and 60 Gr genes through alternative splicing. We include two previously undescribed Or genes and two previously undescribed Gr genes; two previously predicted Or genes are shown to be alternative splice forms. Three polymorphic pseudogenes and one highly defective pseudogene are recognized. Phylogenetic analysis reveals deep branches connecting multiple highly divergent clades within the Gr family, and the Or family appears to be a single highly expanded lineage within the superfamily. The genes are spread throughout the Drosophila genome, with some relatively recently diverged genes still clustered in the genome. The Gr5a gene on the X chromosome, which encodes a receptor for the sugar trehalose, has transposed from one such tandem cluster of six genes at cytological location 64, as has Gr61a, and all eight of these receptors might bind sugars. Analysis of intron evolution suggests that the common ancestor consisted of a long N-terminal exon encoding transmembrane domains 1-5 followed by three exons encoding transmembrane domains 6-7. As many as 57 additional introns have been acquired idiosyncratically during the evolution of the superfamily, whereas the ancestral introns and some of the older idiosyncratic introns have been lost at least 48 times independently. Altogether, these patterns of molecular evolution suggest that this is an ancient superfamily of chemoreceptors, probably dating back at least to the origin of the arthropods. PMID:14608037

Parsing the roles of neck-linker docking and tethered head diffusion in the stepping dynamics of kinesin.

Science.gov (United States)

Zhang, Zhechun; Goldtzvik, Yonathan; Thirumalai, D

2017-11-14

Kinesin walks processively on microtubules (MTs) in an asymmetric hand-over-hand manner consuming one ATP molecule per 16-nm step. The individual contributions due to docking of the approximately 13-residue neck linker to the leading head (deemed to be the power stroke) and diffusion of the trailing head (TH) that contributes in propelling the motor by 16 nm have not been quantified. We use molecular simulations by creating a coarse-grained model of the MT-kinesin complex, which reproduces the measured stall force as well as the force required to dislodge the motor head from the MT, to show that nearly three-quarters of the step occurs by bidirectional stochastic motion of the TH. However, docking of the neck linker to the leading head constrains the extent of diffusion and minimizes the probability that kinesin takes side steps, implying that both the events are necessary in the motility of kinesin and for the maintenance of processivity. Surprisingly, we find that during a single step, the TH stochastically hops multiple times between the geometrically accessible neighboring sites on the MT before forming a stable interaction with the target binding site with correct orientation between the motor head and the [Formula: see text] tubulin dimer.

Crystallization and X-ray diffraction analysis of the CH domain of the cotton kinesin GhKCH2

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Qin, Xinghua [China Agricultural University, No. 2 Yuanmingyuanxilu, Haidian District, Beijing 100094, People’s Republic of (China); The Fourth Military Medical University, No. 169 Changlexi Road, Xincheng District, Xi’an 710032, People’s Republic of (China); Chen, Ziwei; Li, Ping; Liu, Guoqin, E-mail: liu@cau.edu.cn [China Agricultural University, No. 2 Yuanmingyuanxilu, Haidian District, Beijing 100094, People’s Republic of (China)

2016-02-19

The cloning, expression, purification and crystallization of the CH domain of the plant-specific kinesin GhKCH2 is reported. GhKCH2 belongs to a group of plant-specific kinesins (KCHs) containing an actin-binding calponin homology (CH) domain in the N-terminus. Previous studies revealed that the GhKCH2 CH domain (GhKCH2-CH) had a higher affinity for F-actin (K{sub d} = 0.42 ± 0.02 µM) than most other CH-domain-containing proteins. To understand the underlying mechanism, prokaryotically expressed GhKCH2-CH (amino acids 30–166) was purified and crystallized. Crystals were grown by the sitting-drop vapour-diffusion method using 0.1 M Tris–HCl pH 7.0, 20%(w/v) PEG 8000 as a precipitant. The crystals diffracted to a resolution of 2.5 Å and belonged to space group P2{sub 1}, with unit-cell parameters a = 41.57, b = 81.92, c = 83.00 Å, α = 90.00, β = 97.31, γ = 90.00°. Four molecules were found in the asymmetric unit with a Matthews coefficient of 2.22 Å{sup 3} Da{sup −1}, corresponding to a solvent content of 44.8%.

Evolution and Diversity of the Ras Superfamily of Small GTPases in Prokaryotes

Science.gov (United States)

Wuichet, Kristin; Søgaard-Andersen, Lotte

2015-01-01

The Ras superfamily of small GTPases are single domain nucleotide-dependent molecular switches that act as highly tuned regulators of complex signal transduction pathways. Originally identified in eukaryotes for their roles in fundamental cellular processes including proliferation, motility, polarity, nuclear transport, and vesicle transport, recent studies have revealed that single domain GTPases also control complex functions such as cell polarity, motility, predation, development and antibiotic resistance in bacteria. Here, we used a computational genomics approach to understand the abundance, diversity, and evolution of small GTPases in prokaryotes. We collected 520 small GTPase sequences present in 17% of 1,611 prokaryotic genomes analyzed that cover diverse lineages. We identified two discrete families of small GTPases in prokaryotes that show evidence of three distinct catalytic mechanisms. The MglA family includes MglA homologs, which are typically associated with the MglB GTPase activating protein, whereas members of the Rup (Ras superfamily GTPase of unknown function in prokaryotes) family are not predicted to interact with MglB homologs. System classification and genome context analyses support the involvement of small GTPases in diverse prokaryotic signal transduction pathways including two component systems, laying the foundation for future experimental characterization of these proteins. Phylogenetic analysis of prokaryotic and eukaryotic GTPases supports that the last universal common ancestor contained ancestral MglA and Rup family members. We propose that the MglA family was lost from the ancestral eukaryote and that the Ras superfamily members in extant eukaryotes are the result of vertical and horizontal gene transfer events of ancestral Rup GTPases. PMID:25480683

CD147 Immunoglobulin Superfamily Receptor Function and Role in Pathology

OpenAIRE

Iacono, Kathryn T.; Brown, Amy L.; Greene, Mark I.; Saouaf, Sandra J.

2007-01-01

The immunoglobulin superfamily member CD147 plays an important role in fetal, neuronal, lymphocyte and extracellular matrix development. Here we review the current understanding of CD147 expression and protein interactions with regard to CD147 function and its role in pathologic conditions including heart disease, Alzheimer’s disease, stroke and cancer. A model linking hypoxic conditions found within the tumor microenvironment to up-regulation of CD147 expression and tumor progression is intr...

Role of kinesin heavy chain in Crumbs localization along the rhabdomere elongation in Drosophila photoreceptor.

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Garrett P League

Full Text Available BACKGROUND: Crumbs (Crb, a cell polarity gene, has been shown to provide a positional cue for the extension of the apical membrane domain, adherens junction (AJ, and rhabdomere along the growing proximal-distal axis during Drosophila photoreceptor morphogenesis. In developing Drosophila photoreceptors, a stabilized microtubule structure was discovered and its presence was linked to polarity protein localization. It was therefore hypothesized that the microtubules may provide trafficking routes for the polarity proteins during photoreceptor morphogenesis. This study has examined whether Kinesin heavy chain (Khc, a subunit of the microtubule-based motor Kinesin-1, is essential in polarity protein localization in developing photoreceptors. METHODOLOGY/PRINCIPAL FINDINGS: Because a genetic interaction was found between crb and khc, Crb localization was examined in the developing photoreceptors of khc mutants. khc was dispensable during early eye differentiation and development. However, khc mutant photoreceptors showed a range of abnormalities in the apical membrane domain depending on the position along the proximal-distal axis in pupal photoreceptors. The khc mutant showed a progressive mislocalization in the apical domain along the distal-proximal axis during rhabdomere elongation. The khc mutation also led to a similar progressive defect in the stabilized microtubule structures, strongly suggesting that Khc is essential for microtubule structure and Crb localization during distal to proximal rhabdomere elongation in pupal morphogenesis. This role of Khc in apical domain control was further supported by khc's gain-of-function phenotype. Khc overexpression in photoreceptors caused disruption of the apical membrane domain and the stabilized microtubules in the developing photoreceptors. CONCLUSIONS/SIGNIFICANCE: In summary, we examined the role of khc in the regulation of the apical Crb domain in developing photoreceptors. Since the rhabdomeres in

Solution structure and phylogenetics of Prod1, a member of the three-finger protein superfamily implicated in salamander limb regeneration.

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Acely Garza-Garcia

Full Text Available BACKGROUND: Following the amputation of a limb, newts and salamanders have the capability to regenerate the lost tissues via a complex process that takes place at the site of injury. Initially these cells undergo dedifferentiation to a state competent to regenerate the missing limb structures. Crucially, dedifferentiated cells have memory of their level of origin along the proximodistal (PD axis of the limb, a property known as positional identity. Notophthalmus viridescens Prod1 is a cell-surface molecule of the three-finger protein (TFP superfamily involved in the specification of newt limb PD identity. The TFP superfamily is a highly diverse group of metazoan proteins that includes snake venom toxins, mammalian transmembrane receptors and miscellaneous signaling molecules. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of identifying potential orthologs of Prod1, we have solved its 3D structure and compared it to other known TFPs using phylogenetic techniques. The analysis shows that TFP 3D structures group in different categories according to function. Prod1 clusters with other cell surface protein TFP domains including the complement regulator CD59 and the C-terminal domain of urokinase-type plasminogen activator. To infer orthology, a structure-based multiple sequence alignment of representative TFP family members was built and analyzed by phylogenetic methods. Prod1 has been proposed to be the salamander CD59 but our analysis fails to support this association. Prod1 is not a good match for any of the TFP families present in mammals and this result was further supported by the identification of the putative orthologs of both CD59 and N. viridescens Prod1 in sequence data for the salamander Ambystoma tigrinum. CONCLUSIONS/SIGNIFICANCE: The available data suggest that Prod1, and thereby its role in encoding PD identity, is restricted to salamanders. The lack of comparable limb-regenerative capability in other adult vertebrates could be

Two differentially regulated Arabidopsis genes define a new branch of the DFR superfamily

DEFF Research Database (Denmark)

Østergaard, L; Lauvergeat, V; Naested, H

2001-01-01

that, whereas high expression of AtCRL1 in mature seeds declines during subsequent vegetative growth, transcriptional activity from the AtCRL2 promoter increases during vegetative growth. Expression of both genes is restricted to vascular tissue. Based upon their homology to proteins involved in lignin......Two tandem genes were identified on Arabidopsis chromosome II (AtCRL1 and AtCRL2) encoding proteins with homology to members of the dihydroflavonol-4-reductase (DFR) superfamily. The encoded CRL1 and CRL2 proteins share 87% mutual amino acid sequence identity whereas their promoter regions...

Kinesin-2 KIF3AB exhibits novel ATPase characteristics.

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Albracht, Clayton D; Rank, Katherine C; Obrzut, Steven; Rayment, Ivan; Gilbert, Susan P

2014-10-03

KIF3AB is an N-terminal processive kinesin-2 family member best known for its role in intraflagellar transport. There has been significant interest in KIF3AB in defining the key principles that underlie the processivity of KIF3AB in comparison with homodimeric processive kinesins. To define the ATPase mechanism and coordination of KIF3A and KIF3B stepping, a presteady-state kinetic analysis was pursued. For these studies, a truncated murine KIF3AB was generated. The results presented show that microtubule association was fast at 5.7 μm(-1) s(-1), followed by rate-limiting ADP release at 12.8 s(-1). ATP binding at 7.5 μm(-1) s(-1) was followed by an ATP-promoted isomerization at 84 s(-1) to form the intermediate poised for ATP hydrolysis, which then occurred at 33 s(-1). ATP hydrolysis was required for dissociation of the microtubule·KIF3AB complex, which was observed at 22 s(-1). The dissociation step showed an apparent affinity for ATP that was very weak (K½,ATP at 133 μm). Moreover, the linear fit of the initial ATP concentration dependence of the dissociation kinetics revealed an apparent second-order rate constant at 0.09 μm(-1) s(-1), which is inconsistent with fast ATP binding at 7.5 μm(-1) s(-1) and a Kd ,ATP at 6.1 μm. These results suggest that ATP binding per se cannot account for the apparent weak K½,ATP at 133 μm. The steady-state ATPase Km ,ATP, as well as the dissociation kinetics, reveal an unusual property of KIF3AB that is not yet well understood and also suggests that the mechanochemistry of KIF3AB is tuned somewhat differently from homodimeric processive kinesins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A SNARE-Like Superfamily Protein SbSLSP from the Halophyte Salicornia brachiata Confers Salt and Drought Tolerance by Maintaining Membrane Stability, K(+)/Na(+) Ratio, and Antioxidant Machinery.

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Singh, Dinkar; Yadav, Narendra Singh; Tiwari, Vivekanand; Agarwal, Pradeep K; Jha, Bhavanath

2016-01-01

About 1000 salt-responsive ESTs were identified from an extreme halophyte Salicornia brachiata. Among these, a novel salt-inducible gene SbSLSP (Salicornia brachiata SNARE-like superfamily protein), showed up-regulation upon salinity and dehydration stress. The presence of cis-regulatory motifs related to abiotic stress in the putative promoter region supports our finding that SbSLSP gene is inducible by abiotic stress. The SbSLSP protein showed a high sequence identity to hypothetical/uncharacterized proteins from Beta vulgaris, Spinacia oleracea, Eucalyptus grandis, and Prunus persica and with SNARE-like superfamily proteins from Zostera marina and Arabidopsis thaliana. Bioinformatics analysis predicted a clathrin adaptor complex small-chain domain and N-myristoylation site in the SbSLSP protein. Subcellular localization studies indicated that the SbSLSP protein is mainly localized in the plasma membrane. Using transgenic tobacco lines, we establish that overexpression of SbSLSP resulted in elevated tolerance to salt and drought stress. The improved tolerance was confirmed by alterations in a range of physiological parameters, including high germination and survival rate, higher leaf chlorophyll contents, and reduced accumulation of Na(+) ion and reactive oxygen species (ROS). Furthermore, overexpressing lines also showed lower water loss, higher cell membrane stability, and increased accumulation of proline and ROS-scavenging enzymes. Overexpression of SbSLSP also enhanced the transcript levels of ROS-scavenging and signaling enzyme genes. This study is the first investigation of the function of the SbSLSP gene as a novel determinant of salinity/drought tolerance. The results suggest that SbSLSP could be a potential candidate to increase salinity and drought tolerance in crop plants for sustainable agriculture in semi-arid saline soil.

A SNARE-like superfamily protein SbSLSP from the halophyte Salicornia brachiata confers salt and drought tolerance by maintaining membrane stability, K+/Na+ ratio, and antioxidant machinery

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Dinkar eSingh

2016-06-01

Full Text Available About 1000 salt-responsive ESTs were identified from an extreme halophyte Salicornia brachiata. Among these, a novel salt-inducible gene SbSLSP, (Salicornia brachiata SNARE-like superfamily protein showed up-regulation upon salinity and dehydration stress. The presence of cis-regulatory motifs related to abiotic stress in the putative promoter region supports our finding that SbSLSP gene is inducible by abiotic stress. The SbSLSP protein showed a high sequence identity to hypothetical/uncharacterised proteins from Beta vulgaris, Spinacia oleracea, Eucalyptus grandis and Prunus persica and with SNARE-like superfamily proteins from Zostera marina and Arabidopsis thaliana. Bioinformatics analysis predicted a clathrin adaptor complex small-chain domain and N-myristoylation site in the SbSLSP protein. Subcellular localisation studies indicated that the SbSLSP protein is mainly localised in the plasma membrane. Using transgenic tobacco lines, we establish that overexpression of SbSLSP resulted in elevated tolerance to salt and drought stress. The improved tolerance was confirmed by alterations in a range of physiological parameters, including high germination and survival rate, higher leaf chlorophyll contents, and reduced accumulation of Na+ ion and reactive oxygen species (ROS. Furthermore, overexpressing lines also showed lower water loss, higher cell membrane stability and increased accumulation of proline and ROS-scavenging enzymes. Overexpression of SbSLSP also enhanced the transcript levels of ROS-scavenging and signalling enzyme genes. This study is the first investigation of the function of the SbSLSP gene as a novel determinant of salinity/drought tolerance. The results suggest that SbSLSP could be a potential candidate to increase salinity and drought tolerance in crop plants for sustainable agriculture in semi-arid saline soil.

Cell Adhesion Molecules of the Immunoglobulin Superfamily in the Nervous System

DEFF Research Database (Denmark)

Walmod, Peter Schledermann; Pedersen, Martin Volmer; Berezin, Vladimir

2007-01-01

Cell adhesion molecules (CAMs) are proteins mediating cell-cell or cell-extracellular matrix (ECM) interactions. CAMs are traditionally divided into four groups, the cadherins, the selectins, the integrins and CAMs belonging to the immunoglobulin superfamily (IgSF). The present chapter describes...... CAMs belonging to IgSF, that exclusively or in part, are expressed in the nervous system. The chapter includes descriptions of myelin protein zero (P0), integrin-associated protein (CD47), neuroplastin, activated leukocyte-cell adhesion molecule (ALCAM), melanoma cell adhesion molecule (MCAM......), myelinassociated glycoprotein (MAG), the neural cell adhesion molecules 1 and 2 (NCAM, NCAM2), Down Syndrome cell adhesion molecule (DSCAM) and Down Syndrome cell adhesion molecule-like-1 (DSCAML1), sidekick 1 and 2 (SDK1, SDK2), signal-regulatory proteins (SIRPs), nectins, nectin-like proteins (necls...

In-silico gene co-expression network analysis in Paracoccidioides brasiliensis with reference to haloacid dehalogenase superfamily hydrolase gene

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Raghunath Satpathy

2015-01-01

Full Text Available Context: Paracoccidioides brasiliensis, a dimorphic fungus is the causative agent of paracoccidioidomycosis, a disease globally affecting millions of people. The haloacid dehalogenase (HAD superfamily hydrolases enzyme in the fungi, in particular, is known to be responsible in the pathogenesis by adhering to the tissue. Hence, identification of novel drug targets is essential. Aims: In-silico based identification of co-expressed genes along with HAD superfamily hydrolase in P. brasiliensis during the morphogenesis from mycelium to yeast to identify possible genes as drug targets. Materials and Methods: In total, four datasets were retrieved from the NCBI-gene expression omnibus (GEO database, each containing 4340 genes, followed by gene filtration expression of the data set. Further co-expression (CE study was performed individually and then a combination these genes were visualized in the Cytoscape 2. 8.3. Statistical Analysis Used: Mean and standard deviation value of the HAD superfamily hydrolase gene was obtained from the expression data and this value was subsequently used for the CE calculation purpose by selecting specific correlation power and filtering threshold. Results: The 23 genes that were thus obtained are common with respect to the HAD superfamily hydrolase gene. A significant network was selected from the Cytoscape network visualization that contains total 7 genes out of which 5 genes, which do not have significant protein hits, obtained from gene annotation of the expressed sequence tags by BLAST X. For all the protein PSI-BLAST was performed against human genome to find the homology. Conclusions: The gene co-expression network was obtained with respect to HAD superfamily dehalogenase gene in P. Brasiliensis.

RECEPTOR SUPERFAMILY OF TUMOR NECROSIS FACTOR Α, AND HSP90 HEAT SHOCK PROTEIN: A MOLECULAR BASIS FOR INTERACTIONS

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N. V. Ryazantseva

2011-01-01

Full Text Available Abstract.  A  study  was  performed  aiming  to  investigate  interactions  between  TNFα  receptor  (TNF1 superfamily and heat shock protein Hsp90, using a Jurkat tumor cell line. The tumor cells cultured in presence of Hsp90 inhibitor (17-AAG showed increased numbers of cells, presenting surface TNFR1 and FasR, which facilitate  triggering  of  programmed  cell  death.  It  was  also  revealed  that  Hsp90  blockage  under  the  in  vitro conditions causes a decrease in FasL, while not affecting TNFα and sTNFR1 production by the tumor cells. (Med. Immunol., 2011, vol. 13, N 2-3, pp 247-252 

Unc-51/ATG1 controls axonal and dendritic development via kinesin-mediated vesicle transport in the Drosophila brain.

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Hiroaki Mochizuki

2011-05-01

Full Text Available Members of the evolutionary conserved Ser/Thr kinase Unc-51 family are key regulatory proteins that control neural development in both vertebrates and invertebrates. Previous studies have suggested diverse functions for the Unc-51 protein, including axonal elongation, growth cone guidance, and synaptic vesicle transport.In this work, we have investigated the functional significance of Unc-51-mediated vesicle transport in the development of complex brain structures in Drosophila. We show that Unc-51 preferentially accumulates in newly elongating axons of the mushroom body, a center of olfactory learning in flies. Mutations in unc-51 cause disintegration of the core of the developing mushroom body, with mislocalization of Fasciclin II (Fas II, an IgG-family cell adhesion molecule important for axonal guidance and fasciculation. In unc-51 mutants, Fas II accumulates in the cell bodies, calyx, and the proximal peduncle. Furthermore, we show that mutations in unc-51 cause aberrant overshooting of dendrites in the mushroom body and the antennal lobe. Loss of unc-51 function leads to marked accumulation of Rab5 and Golgi components, whereas the localization of dendrite-specific proteins, such as Down syndrome cell adhesion molecule (DSCAM and No distributive disjunction (Nod, remains unaltered. Genetic analyses of kinesin light chain (Klc and unc-51 double heterozygotes suggest the importance of kinesin-mediated membrane transport for axonal and dendritic development. Moreover, our data demonstrate that loss of Klc activity causes similar axonal and dendritic defects in mushroom body neurons, recapitulating the salient feature of the developmental abnormalities caused by unc-51 mutations.Unc-51 plays pivotal roles in the axonal and dendritic development of the Drosophila brain. Unc-51-mediated membrane vesicle transport is important in targeted localization of guidance molecules and organelles that regulate elongation and compartmentalization of

Subdivision of the MDR superfamily of medium-chain dehydrogenases/reductases through iterative hidden Markov model refinement

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Persson Bengt

2010-10-01

Full Text Available Abstract Background The Medium-chain Dehydrogenases/Reductases (MDR form a protein superfamily whose size and complexity defeats traditional means of subclassification; it currently has over 15000 members in the databases, the pairwise sequence identity is typically around 25%, there are members from all kingdoms of life, the chain-lengths vary as does the oligomericity, and the members are partaking in a multitude of biological processes. There are profile hidden Markov models (HMMs available for detecting MDR superfamily members, but none for determining which MDR family each protein belongs to. The current torrential influx of new sequence data enables elucidation of more and more protein families, and at an increasingly fine granularity. However, gathering good quality training data usually requires manual attention by experts and has therefore been the rate limiting step for expanding the number of available models. Results We have developed an automated algorithm for HMM refinement that produces stable and reliable models for protein families. This algorithm uses relationships found in data to generate confident seed sets. Using this algorithm we have produced HMMs for 86 distinct MDR families and 34 of their subfamilies which can be used in automated annotation of new sequences. We find that MDR forms with 2 Zn2+ ions in general are dehydrogenases, while MDR forms with no Zn2+ in general are reductases. Furthermore, in Bacteria MDRs without Zn2+ are more frequent than those with Zn2+, while the opposite is true for eukaryotic MDRs, indicating that Zn2+ has been recruited into the MDR superfamily after the initial life kingdom separations. We have also developed a web site http://mdr-enzymes.org that provides textual and numeric search against various characterised MDR family properties, as well as sequence scan functions for reliable classification of novel MDR sequences. Conclusions Our method of refinement can be readily applied to

Evolutionarily conserved substrate substructures for automated annotation of enzyme superfamilies.

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Ranyee A Chiang

2008-08-01

Full Text Available The evolution of enzymes affects how well a species can adapt to new environmental conditions. During enzyme evolution, certain aspects of molecular function are conserved while other aspects can vary. Aspects of function that are more difficult to change or that need to be reused in multiple contexts are often conserved, while those that vary may indicate functions that are more easily changed or that are no longer required. In analogy to the study of conservation patterns in enzyme sequences and structures, we have examined the patterns of conservation and variation in enzyme function by analyzing graph isomorphisms among enzyme substrates of a large number of enzyme superfamilies. This systematic analysis of substrate substructures establishes the conservation patterns that typify individual superfamilies. Specifically, we determined the chemical substructures that are conserved among all known substrates of a superfamily and the substructures that are reacting in these substrates and then examined the relationship between the two. Across the 42 superfamilies that were analyzed, substantial variation was found in how much of the conserved substructure is reacting, suggesting that superfamilies may not be easily grouped into discrete and separable categories. Instead, our results suggest that many superfamilies may need to be treated individually for analyses of evolution, function prediction, and guiding enzyme engineering strategies. Annotating superfamilies with these conserved and reacting substructure patterns provides information that is orthogonal to information provided by studies of conservation in superfamily sequences and structures, thereby improving the precision with which we can predict the functions of enzymes of unknown function and direct studies in enzyme engineering. Because the method is automated, it is suitable for large-scale characterization and comparison of fundamental functional capabilities of both characterized

Evolutionarily conserved substrate substructures for automated annotation of enzyme superfamilies.

Science.gov (United States)

Chiang, Ranyee A; Sali, Andrej; Babbitt, Patricia C

2008-08-01

The evolution of enzymes affects how well a species can adapt to new environmental conditions. During enzyme evolution, certain aspects of molecular function are conserved while other aspects can vary. Aspects of function that are more difficult to change or that need to be reused in multiple contexts are often conserved, while those that vary may indicate functions that are more easily changed or that are no longer required. In analogy to the study of conservation patterns in enzyme sequences and structures, we have examined the patterns of conservation and variation in enzyme function by analyzing graph isomorphisms among enzyme substrates of a large number of enzyme superfamilies. This systematic analysis of substrate substructures establishes the conservation patterns that typify individual superfamilies. Specifically, we determined the chemical substructures that are conserved among all known substrates of a superfamily and the substructures that are reacting in these substrates and then examined the relationship between the two. Across the 42 superfamilies that were analyzed, substantial variation was found in how much of the conserved substructure is reacting, suggesting that superfamilies may not be easily grouped into discrete and separable categories. Instead, our results suggest that many superfamilies may need to be treated individually for analyses of evolution, function prediction, and guiding enzyme engineering strategies. Annotating superfamilies with these conserved and reacting substructure patterns provides information that is orthogonal to information provided by studies of conservation in superfamily sequences and structures, thereby improving the precision with which we can predict the functions of enzymes of unknown function and direct studies in enzyme engineering. Because the method is automated, it is suitable for large-scale characterization and comparison of fundamental functional capabilities of both characterized and uncharacterized

Characterizing and modeling protein-surface interactions in lab-on-chip devices

Science.gov (United States)

Katira, Parag

Protein adsorption on surfaces determines the response of other biological species present in the surrounding solution. This phenomenon plays a major role in the design of biomedical and biotechnological devices. While specific protein adsorption is essential for device function, non-specific protein adsorption leads to the loss of device function. For example, non-specific protein adsorption on bioimplants triggers foreign body response, in biosensors it leads to reduced signal to noise ratios, and in hybrid bionanodevices it results in the loss of confinement and directionality of molecular shuttles. Novel surface coatings are being developed to reduce or completely prevent the non-specific adsorption of proteins to surfaces. A novel quantification technique for extremely low protein coverage on surfaces has been developed. This technique utilizes measurement of the landing rate of microtubule filaments on kinesin proteins adsorbed on a surface to determine the kinesin density. Ultra-low limits of detection, dynamic range, ease of detection and availability of a ready-made kinesin-microtubule kit makes this technique highly suitable for detecting protein adsorption below the detection limits of standard techniques. Secondly, a random sequential adsorption model is presented for protein adsorption to PEO-coated surfaces. The derived analytical expressions accurately predict the observed experimental results from various research groups, suggesting that PEO chains act as almost perfect steric barriers to protein adsorption. These expressions can be used to predict the performance of a variety of systems towards resisting protein adsorption and can help in the design of better non-fouling surface coatings. Finally, in biosensing systems, target analytes are captured and concentrated on specifically adsorbed proteins for detection. Non-specific adsorption of proteins results in the loss of signal, and an increase in the background. The use of nanoscale transducers as

Stability for Function Trade-Offs in the Enolase Superfamily 'Catalytic Module'

Energy Technology Data Exchange (ETDEWEB)

Nagatani, R.A.; Gonzalez, A.; Shoichet, B.K.; Brinen, L.S.; Babbitt, P.C.; /UC, San Francisco /SLAC, SSRL

2007-07-12

Enzyme catalysis reflects a dynamic interplay between charged and polar active site residues that facilitate function, stabilize transition states, and maintain overall protein stability. Previous studies show that substituting neutral for charged residues in the active site often significantly stabilizes a protein, suggesting a stability trade-off for functionality. In the enolase superfamily, a set of conserved active site residues (the ''catalytic module'') has repeatedly been used in nature in the evolution of many different enzymes for the performance of unique overall reactions involving a chemically diverse set of substrates. This catalytic module provides a robust solution for catalysis that delivers the common underlying partial reaction that supports all of the different overall chemical reactions of the superfamily. As this module has been so broadly conserved in the evolution of new functions, we sought to investigate the extent to which it follows the stability-function trade-off. Alanine substitutions were made for individual residues, groups of residues, and the entire catalytic module of o-succinylbenzoate synthase (OSBS), a member of the enolase superfamily from Escherichia coli. Of six individual residue substitutions, four (K131A, D161A, E190A, and D213A) substantially increased protein stability (by 0.46-4.23 kcal/mol), broadly consistent with prediction of a stability-activity trade-off. The residue most conserved across the superfamily, E190, is by far the most destabilizing. When the individual substitutions were combined into groups (as they are structurally and functionally organized), nonadditive stability effects emerged, supporting previous observations that residues within the module interact as two functional groups within a larger catalytic system. Thus, whereas the multiple-mutant enzymes D161A/E190A/D213A and K131A/K133A/D161A/E190A/D213A/K235A (termed 3KDED) are stabilized relative to the wild-type enzyme (by 1

Annotation error in public databases: misannotation of molecular function in enzyme superfamilies.

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Alexandra M Schnoes

2009-12-01

Full Text Available Due to the rapid release of new data from genome sequencing projects, the majority of protein sequences in public databases have not been experimentally characterized; rather, sequences are annotated using computational analysis. The level of misannotation and the types of misannotation in large public databases are currently unknown and have not been analyzed in depth. We have investigated the misannotation levels for molecular function in four public protein sequence databases (UniProtKB/Swiss-Prot, GenBank NR, UniProtKB/TrEMBL, and KEGG for a model set of 37 enzyme families for which extensive experimental information is available. The manually curated database Swiss-Prot shows the lowest annotation error levels (close to 0% for most families; the two other protein sequence databases (GenBank NR and TrEMBL and the protein sequences in the KEGG pathways database exhibit similar and surprisingly high levels of misannotation that average 5%-63% across the six superfamilies studied. For 10 of the 37 families examined, the level of misannotation in one or more of these databases is >80%. Examination of the NR database over time shows that misannotation has increased from 1993 to 2005. The types of misannotation that were found fall into several categories, most associated with "overprediction" of molecular function. These results suggest that misannotation in enzyme superfamilies containing multiple families that catalyze different reactions is a larger problem than has been recognized. Strategies are suggested for addressing some of the systematic problems contributing to these high levels of misannotation.

Annotation error in public databases: misannotation of molecular function in enzyme superfamilies.

Science.gov (United States)

Schnoes, Alexandra M; Brown, Shoshana D; Dodevski, Igor; Babbitt, Patricia C

2009-12-01

Due to the rapid release of new data from genome sequencing projects, the majority of protein sequences in public databases have not been experimentally characterized; rather, sequences are annotated using computational analysis. The level of misannotation and the types of misannotation in large public databases are currently unknown and have not been analyzed in depth. We have investigated the misannotation levels for molecular function in four public protein sequence databases (UniProtKB/Swiss-Prot, GenBank NR, UniProtKB/TrEMBL, and KEGG) for a model set of 37 enzyme families for which extensive experimental information is available. The manually curated database Swiss-Prot shows the lowest annotation error levels (close to 0% for most families); the two other protein sequence databases (GenBank NR and TrEMBL) and the protein sequences in the KEGG pathways database exhibit similar and surprisingly high levels of misannotation that average 5%-63% across the six superfamilies studied. For 10 of the 37 families examined, the level of misannotation in one or more of these databases is >80%. Examination of the NR database over time shows that misannotation has increased from 1993 to 2005. The types of misannotation that were found fall into several categories, most associated with "overprediction" of molecular function. These results suggest that misannotation in enzyme superfamilies containing multiple families that catalyze different reactions is a larger problem than has been recognized. Strategies are suggested for addressing some of the systematic problems contributing to these high levels of misannotation.

Main trends of karyotype evolution in the superfamily Chalcidoidea (Hymenoptera

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Vladimir Gokhman

2009-08-01

Full Text Available An overview of karyotype evolution in the superfamily Chalcidoidea is given. Structural types of chromosome sets in the superfamily are listed. Main pathways of karyotypic change in the Chalcidoidea are outlined. The chromosome set containing eleven subtelo- or acrocentrics is considered as an ancestral karyotype for the superfamily. Multiple independent reductions in n values through chromosomal fusions presumably occurred in various groups of chalcid families.

Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae: BIOCHEMICAL, STRUCTURAL, AND EVOLUTIONARY INSIGHTS.

Science.gov (United States)

Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S; Flick, Robert; Wolf, Yuri I; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M; Koonin, Eugene V; Yakunin, Alexander F

2015-07-24

The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cytoplasmic streaming in Drosophila oocytes varies with kinesin activity and correlates with the microtubule cytoskeleton architecture.

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Ganguly, Sujoy; Williams, Lucy S; Palacios, Isabel M; Goldstein, Raymond E

2012-09-18

Cells can localize molecules asymmetrically through the combined action of cytoplasmic streaming, which circulates their fluid contents, and specific anchoring mechanisms. Streaming also contributes to the distribution of nutrients and organelles such as chloroplasts in plants, the asymmetric position of the meiotic spindle in mammalian embryos, and the developmental potential of the zygote, yet little is known quantitatively about the relationship between streaming and the motor activity which drives it. Here we use Particle Image Velocimetry to quantify the statistical properties of Kinesin-dependent streaming during mid-oogenesis in Drosophila. We find that streaming can be used to detect subtle changes in Kinesin activity and that the flows reflect the architecture of the microtubule cytoskeleton. Furthermore, based on characterization of the rheology of the cytoplasm in vivo, we establish estimates of the number of Kinesins required to drive the observed streaming. Using this in vivo data as the basis of a model for transport, we suggest that the disordered character of transport at mid-oogenesis, as revealed by streaming, is an important component of the localization dynamics of the body plan determinant oskar mRNA.

A Mechanism for Cytoplasmic Streaming: Kinesin-Driven Alignment of Microtubules and Fast Fluid Flows.

Science.gov (United States)

Monteith, Corey E; Brunner, Matthew E; Djagaeva, Inna; Bielecki, Anthony M; Deutsch, Joshua M; Saxton, William M

2016-05-10

The transport of cytoplasmic components can be profoundly affected by hydrodynamics. Cytoplasmic streaming in Drosophila oocytes offers a striking example. Forces on fluid from kinesin-1 are initially directed by a disordered meshwork of microtubules, generating minor slow cytoplasmic flows. Subsequently, to mix incoming nurse cell cytoplasm with ooplasm, a subcortical layer of microtubules forms parallel arrays that support long-range, fast flows. To analyze the streaming mechanism, we combined observations of microtubule and organelle motions with detailed mathematical modeling. In the fast state, microtubules tethered to the cortex form a thin subcortical layer and undergo correlated sinusoidal bending. Organelles moving in flows along the arrays show velocities that are slow near the cortex and fast on the inward side of the subcortical microtubule layer. Starting with fundamental physical principles suggested by qualitative hypotheses, and with published values for microtubule stiffness, kinesin velocity, and cytoplasmic viscosity, we developed a quantitative coupled hydrodynamic model for streaming. The fully detailed mathematical model and its simulations identify key variables that can shift the system between disordered (slow) and ordered (fast) states. Measurements of array curvature, wave period, and the effects of diminished kinesin velocity on flow rates, as well as prior observations on f-actin perturbation, support the model. This establishes a concrete mechanistic framework for the ooplasmic streaming process. The self-organizing fast phase is a result of viscous drag on kinesin-driven cargoes that mediates equal and opposite forces on cytoplasmic fluid and on microtubules whose minus ends are tethered to the cortex. Fluid moves toward plus ends and microtubules are forced backward toward their minus ends, resulting in buckling. Under certain conditions, the buckling microtubules self-organize into parallel bending arrays, guiding varying directions

Discovery of (+)-N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877), a kinesin spindle protein inhibitor and potential anticancer agent.

Science.gov (United States)

Theoclitou, Maria-Elena; Aquila, Brian; Block, Michael H; Brassil, Patrick J; Castriotta, Lillian; Code, Erin; Collins, Michael P; Davies, Audrey M; Deegan, Tracy; Ezhuthachan, Jayachandran; Filla, Sandra; Freed, Ellen; Hu, Haiqing; Huszar, Dennis; Jayaraman, Muthusamy; Lawson, Deborah; Lewis, Paula M; Nadella, Murali V P; Oza, Vibha; Padmanilayam, Maniyan; Pontz, Timothy; Ronco, Lucienne; Russell, Daniel; Whitston, David; Zheng, Xiaolan

2011-10-13

Structure-activity relationship analysis identified (+)-N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877), from a series of novel kinesin spindle protein (KSP) inhibitors, as exhibiting both excellent biochemical potency and pharmaceutical properties suitable for clinical development. The selected compound arrested cells in mitosis leading to the formation of the monopolar spindle phenotype characteristic of KSP inhibition and induction of cellular death. A favorable pharmacokinetic profile and notable in vivo efficacy supported the selection of this compound as a clinical candidate for the treatment of cancer.

Two different groups of signal sequence in M-superfamily conotoxins.

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Wang, Qi; Jiang, Hui; Han, Yu-Hong; Yuan, Duo-Duo; Chi, Cheng-Wu

2008-04-01

M-superfamily conotoxins can be divided into four branches (M-1, M-2, M-3 and M-4) according to the number of amino acid residues in the third Cys loop. In general, it is widely accepted that the conotoxin signal peptides of each superfamily are strictly conserved. Recently, we cloned six cDNAs of novel M-superfamily conotoxins from Conus leopardus, Conus marmoreus and Conus quercinus, belonging to either M-1 or M-3 branch. These conotoxins, judging from the putative peptide sequences deducted from cDNAs, are rich in acidic residues and share highly conserved signal and pro-peptide region. However, they are quite different from the reported conotoxins of M-2 and M-4 branches even in their signal peptides, which in general are considered highly conserved for each superfamily of conotoxins. The signal sequences of M-1 and M-3 conotoxins composed of 24 residues start with MLKMGVVL-, while those of M-2 and M-4 conotoxins composed of 25 residues start with MMSKLGVL-. It is another example that different types of signal peptides can exist within a superfamily besides the I-conotoxin superfamily. In addition to the different disulfide connectivity of M-1 conotoxins from that of M-4 or M-2 conotoxins, the sequence alignment, preferential Cys codon usage and phylogenetic tree analysis suggest that M-1 and M-3 conotoxins have much closer relationship, being different from the conotoxins of other two branches (M-4 and M-2) of M-superfamily.

Immunoglobulin superfamily members encoded by viruses and their multiple roles in immune evasion.

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Farré, Domènec; Martínez-Vicente, Pablo; Engel, Pablo; Angulo, Ana

2017-05-01

Pathogens have developed a plethora of strategies to undermine host immune defenses in order to guarantee their survival. For large DNA viruses, these immune evasion mechanisms frequently rely on the expression of genes acquired from host genomes. Horizontally transferred genes include members of the immunoglobulin superfamily, whose products constitute the most diverse group of proteins of vertebrate genomes. Their promiscuous immunoglobulin domains, which comprise the building blocks of these molecules, are involved in a large variety of functions mediated by ligand-binding interactions. The flexible structural nature of the immunoglobulin domains makes them appealing targets for viral capture due to their capacity to generate high functional diversity. Here, we present an up-to-date review of immunoglobulin superfamily gene homologs encoded by herpesviruses, poxviruses, and adenoviruses, that include CD200, CD47, Fc receptors, interleukin-1 receptor 2, interleukin-18 binding protein, CD80, carcinoembryonic antigen-related cell adhesion molecules, and signaling lymphocyte activation molecules. We discuss their distinct structural attributes, binding properties, and functions, shaped by evolutionary pressures to disarm specific immune pathways. We include several novel genes identified from extensive genome database surveys. An understanding of the properties and modes of action of these viral proteins may guide the development of novel immune-modulatory therapeutic tools. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pulmonary artery hypertension in childhood: The transforming growth factor-β superfamily-related genes

Directory of Open Access Journals (Sweden)

Shi-Min Yuan

2018-04-01

Full Text Available Pulmonary artery hypertension (PAH is very rare in childhood, and it can be divided into heritable, idiopathic drug- and toxin-induced and other disease (connective tissue disease, human immunodeficiency virus infection, portal hypertension, congenital heart disease, or schistosomiasis-associated types. PAH could not be interpreted solely by pathophysiological theories. The impact of the transforming growth factor-β superfamily-related genes on the development of PAH in children remains to be clarified. Pertinent literature on the transforming growth factor-β superfamily-related genes in relation to PAH in children published after the year 2000 was reviewed and analyzed. Bone morphogenetic protein receptor type II gene mutation promotes cell division or prevents cell death, resulting in an overgrowth of cells in small arteries throughout the lungs. About 20% of individuals with a bone morphogenetic protein receptor type II gene mutation develop symptomatic PAH. In heritable PAH, bone morphogenetic protein receptor type II mutations may be absent; while mutations of other genes, such as type I receptor activin receptor-like kinase 1 and the type III receptor endoglin (both associated with hereditary hemorrhagic telangiectasia, caveolin-1 and KCNK3, the gene encoding potassium channel subfamily K, member 3, can be detected, instead. Gene mutations, environmental changes and acquired adjustment, etc. may explain the development of PAH. The researches on PAH rat model and familial PAH members may facilitate the elucidations of the mechanisms and further provide theories for prophylaxis and treatment of PAH. Key Words: bone morphogenetic proteins, mutation, pulmonary hypertension

Superfamily of genes encoding G protein-coupled receptors in the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae).

Science.gov (United States)

Wu, S-F; Yu, H-Y; Jiang, T-T; Gao, C-F; Shen, J-L

2015-08-01

G protein-coupled receptors (GPCRs) are the largest and most versatile superfamily of cell membrane proteins, which mediate various physiological processes including reproduction, development and behaviour. The diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), is one of the most notorious insect pests, preferentially feeding on cruciferous plants. P. xylostella is not only one of the world's most widespread lepidopteran insects, but has also developed resistance to nearly all classes of insecticides. Although the mechanisms of insecticide resistance have been studied extensively in many insect species, few investigations have been carried out on GPCRs in P. xylostella. In the present study, we identified 95 putative GPCRs in the P. xylostella genome. The identified GPCRs were compared with their homologues in Bombyx mori and Drosophila melanogaster. Our results suggest that GPCRs in different insect species may have evolved by a birth-and-death process. One of the differences among compared insects is the duplication of short neuropeptide F receptor and adipokinetic hormone receptors in P. xylostella and B. mori. Another divergence is the decrease in quantity and diversity of the stress-tolerance gene, Mth, in P. xylostella. The evolution by the birth-and-death process is probably involved in adaptation to the feeding behaviour, reproduction and stress responses of P. xylostella. Some of the genes identified in the present study could be potential targets for the development of novel pesticides. © 2015 The Royal Entomological Society.

Exploring the role of cellular homologous of the 30K-superfamily of plant virus movement proteins.

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Carrasco, José L; Sánchez-Navarro, Jesús A; Elena, Santiago F

2018-02-21

Genes orthologous to the 30K-superfamily of movement proteins (MP) from plant viruses have been recently discovered by bioinformatics analyses as integrated elements in the genome of most vascular plants. However, their functional relevance for plants is still unclear. Here, we undertake some preliminary steps into the functional characterization of one of these putative MP genes found in Arabidopsis thaliana. We found that the AtMP gene is expressed at different stages of the plant development, with accumulation being highest in flowers but lowest in mature siliques. We also found down-regulation of the gene may result in a small delay in plant development and in an exacerbation of the negative effect of salinity in germination efficiency. We have also explored whether changes in expression of the endogenous AtMP have any effect on susceptibility to infection with several viruses, and found that the infectivity of tobacco rattle tobravirus was strongly dependent on the expression of the endogenous AtMP. Finally, we have cloned the endogenous MP from four different plant species into an expression vector that allows for specifically assessing their activity as cell-to-cell movement proteins and have shown that though some may still retain the ancestral activity, they do so in a quite inefficient manner, thus suggesting they have acquired a novel function during adaptation to the host genome. Copyright © 2018 Elsevier B.V. All rights reserved.

MetaSINEs: Broad Distribution of a Novel SINE Superfamily in Animals.

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Nishihara, Hidenori; Plazzi, Federico; Passamonti, Marco; Okada, Norihiro

2016-02-12

SINEs (short interspersed elements) are transposable elements that typically originate independently in each taxonomic clade (order/family). However, some SINE families share a highly similar central sequence and are thus categorized as a SINE superfamily. Although only four SINE superfamilies (CORE-SINEs, V-SINEs, DeuSINEs, and Ceph-SINEs) have been reported so far, it is expected that new SINE superfamilies would be discovered by deep exploration of new SINEs in metazoan genomes. Here we describe 15 SINEs, among which 13 are novel, that have a similar 66-bp central region and therefore constitute a new SINE superfamily, MetaSINEs. MetaSINEs are distributed from fish to cnidarians, suggesting their common evolutionary origin at least 640 Ma. Because the 3' tails of MetaSINEs are variable, these SINEs most likely survived by changing their partner long interspersed elements for retrotransposition during evolution. Furthermore, we examined the presence of members of other SINE superfamilies in bivalve genomes and characterized eight new SINEs belonging to the CORE-SINEs, V-SINEs, and DeuSINEs, in addition to the MetaSINEs. The broad distribution of bivalve SINEs suggests that at least three SINEs originated in the common ancestor of Bivalvia. Our comparative analysis of the central domains of the SINEs revealed that, in each superfamily, only a restricted region is shared among all of its members. Because the functions of the central domains of the SINE superfamilies remain unknown, such structural information of SINE superfamilies will be useful for future experimental and comparative analyses to reveal why they have been retained in metazoan genomes during evolution. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Crystallization and preliminary X-ray studies of TON-1713 from Thermococcus onnurineus NA1, a putative member of the haloacid dehalogenase superfamily

International Nuclear Information System (INIS)

Le, Binh Van; Lee, Hyun Sook; Cho, Yona; Kang, Sung Gyun; Kim, Dong Young; Kim, Yang-Gyun; Kim, Kyeong Kyu

2007-01-01

A putative member of the haloacid dehalogenase superfamily from T. onnurineus has been expressed, purified and crystallized using 1.6 M magnesium sulfate as a precipitant. The crystals belonged to the triclinic space group P1 and diffracted to 1.8 Å resolution. The haloacid dehalogenase (HAD) protein superfamily is one of the largest enzyme families and shows hydrolytic activity towards diverse substrates. Structural analyses of enzymes belonging to the HAD family are required to elucidate the molecular basis underlying their broad substrate specificity and reaction mechanism. For this purpose, TON-1713, a hypothetical protein from Thermococcus onnurineus that is a member of the HAD superfamily, was expressed in Escherichia coli, purified and crystallized at 295 K using 1.6 M magnesium sulfate as a precipitant. X-ray diffraction data were collected to 1.8 Å resolution using a synchrotron-radiation source. The crystals belong to the triclinic space group P1, with unit-cell parameters a = 52.5, b = 65.8, c = 203.4 Å, α = 71.1, β = 79.9, γ = 74.3°

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

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

1993-09-01

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

Meeting report - TGF-β superfamily: signaling in development and disease.

Science.gov (United States)

Zhang, Ying E; Newfeld, Stuart J

2013-11-01

The latest advances on the transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) signaling pathways were reported at the July 2013 FASEB Summer Research Conference 'The TGF-β Superfamily: Development and Disease'. The meeting was held in Steamboat Springs, Colorado, USA at 6700 feet above sea level in the Rocky Mountains. This was the seventh biannual meeting in the series. In attendance were investigators from a broad range of disciplines with a common interest in the mechanics of TGF-β and BMP signaling pathways, their normal developmental and homeostatic functions, and the diseases associated with pathway misregulation.

MetaSINEs: Broad Distribution of a Novel SINE Superfamily in Animals

OpenAIRE

Nishihara, Hidenori; Plazzi, Federico; Passamonti, Marco; Okada, Norihiro

2016-01-01

SINEs (short interspersed elements) are transposable elements that typically originate independently in each taxonomic clade (order/family). However, some SINE families share a highly similar central sequence and are thus categorized as a SINE superfamily. Although only four SINE superfamilies (CORE-SINEs, V-SINEs, DeuSINEs, and Ceph-SINEs) have been reported so far, it is expected that new SINE superfamilies would be discovered by deep exploration of new SINEs in metazoan genomes. Here we de...

Clinical value of Xenopus kinesin-like protein 2 as a prognostic marker in patients with digestive system cancers: a systematic review and meta-analysis.

Science.gov (United States)

Wang, Gang; Wang, Qian; Li, Zhengyan; Liu, Chaoxu; He, Xianli

2018-01-01

Xenopus kinesin-like protein 2 (TPX2) is a microtubule-associated protein that plays an important role in spindle assembly and dynamics. However, the clinical and prognostic value of TPX2 in the digestive system cancers remains unclear. The objective of this review was to evaluate the association of TPX2 expression with disease-free survival (DFS), overall survival (OS), and clinicopathological features of digestive system cancers. The software Stata 12.0 was used to analyze the outcomes, including OS, disease-free survival (DFS), and clinicopathological characteristics. A total of 10 eligible studies with 906 patients were included. Elevated TPX2 expression was significantly associated with poor DFS (pooled hazard ratio [HR] =2.48, 95% confidence interval [CI]: 1.96-3.13) and OS (pooled HR =2.66, 95% CI: 2.04-3.48) of digestive system malignancies. Subgroup analyses showed that cancer type, sample size, study quality, and laboratory detection methods did not alter the significant prognostic value of TPX2. Additionally, TPX2 expression was found to be an independent predictive factor for DFS (HR =2.31, 95% CI: 1.78-3.01). TPX2 expression might be associated with TNM stage and pathological grade in digestive system cancer. In conclusion, TPX2 is an independent prognostic factor for survival of patients with digestive system cancer. Furthermore, its overexpression is associated with TNM stage and pathological grade in digestive system cancer.

Comparative analysis of cation/proton antiporter superfamily in plants.

Science.gov (United States)

Ye, Chu-Yu; Yang, Xiaohan; Xia, Xinli; Yin, Weilun

2013-06-01

The cation/proton antiporter superfamily is associated with the transport of monovalent cations across membranes. This superfamily was annotated in the Arabidopsis genome and some members were functionally characterized. In the present study, a systematic analysis of the cation/proton antiporter genes in diverse plant species was reported. We identified 240 cation/proton antiporters in alga, moss, and angiosperm. A phylogenetic tree was constructed showing these 240 members are separated into three families, i.e., Na(+)/H(+) exchangers, K(+) efflux antiporters, and cation/H(+) exchangers. Our analysis revealed that tandem and/or segmental duplications contribute to the expansion of cation/H(+) exchangers in the examined angiosperm species. Sliding window analysis of the nonsynonymous/synonymous substitution ratios showed some differences in the evolutionary fate of cation/proton antiporter paralogs. Furthermore, we identified over-represented motifs among these 240 proteins and found most motifs are family specific, demonstrating diverse evolution of the cation/proton antiporters among three families. In addition, we investigated the co-expressed genes of the cation/proton antiporters in Arabidopsis thaliana. The results showed some biological processes are enriched in the co-expressed genes, suggesting the cation/proton antiporters may be involved in these biological processes. Taken together, this study furthers our knowledge on cation/proton antiporters in plants. Copyright © 2013 Elsevier B.V. All rights reserved.

A global view of structure-function relationships in the tautomerase superfamily.

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Davidson, Rebecca; Baas, Bert-Jan; Akiva, Eyal; Holliday, Gemma L; Polacco, Benjamin J; LeVieux, Jake A; Pullara, Collin R; Zhang, Yan Jessie; Whitman, Christian P; Babbitt, Patricia C

2018-02-16

The tautomerase superfamily (TSF) consists of more than 11,000 nonredundant sequences present throughout the biosphere. Characterized members have attracted much attention because of the unusual and key catalytic role of an N-terminal proline. These few characterized members catalyze a diverse range of chemical reactions, but the full scale of their chemical capabilities and biological functions remains unknown. To gain new insight into TSF structure-function relationships, we performed a global analysis of similarities across the entire superfamily and computed a sequence similarity network to guide classification into distinct subgroups. Our results indicate that TSF members are found in all domains of life, with most being present in bacteria. The eukaryotic members of the cis -3-chloroacrylic acid dehalogenase subgroup are limited to fungal species, whereas the macrophage migration inhibitory factor subgroup has wide eukaryotic representation (including mammals). Unexpectedly, we found that 346 TSF sequences lack Pro-1, of which 85% are present in the malonate semialdehyde decarboxylase subgroup. The computed network also enabled the identification of similarity paths, namely sequences that link functionally diverse subgroups and exhibit transitional structural features that may help explain reaction divergence. A structure-guided comparison of these linker proteins identified conserved transitions between them, and kinetic analysis paralleled these observations. Phylogenetic reconstruction of the linker set was consistent with these findings. Our results also suggest that contemporary TSF members may have evolved from a short 4-oxalocrotonate tautomerase-like ancestor followed by gene duplication and fusion. Our new linker-guided strategy can be used to enrich the discovery of sequence/structure/function transitions in other enzyme superfamilies. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Keanekaragaman Jenis Kupu-Kupu Superfamili Papilionoidae di Banyuwindu, Limbangan Kendal

Directory of Open Access Journals (Sweden)

Ratna Oqtafiana

2013-03-01

Full Text Available Kupu-kupu turut memberi andil dalam mempertahankan keseimbangan ekosistem dan memperkaya keanekaragaman hayati. Tujuan dari penelitian ini adalah untuk mengetahui keanekaragaman jenis kupu-kupu superfamili Papilionoidae di Dukuh Banyuwindu Desa Limbangan Kecamatan Limbangan Kabupaten Kendal khususnya di habitat hutan sekunder, permukiman, Daerah Aliran Sungai (DAS dan persawahan.Populasi dalam penelitian ini adalah semua jenis kupu-kupu superfamili Papilionoidae yang ada di Banyuwindu, Limbangan Kendal. Sampel penelitian ini adalah jenis kupu-kupu superfamili Papilionoidae yang teramati di Banyuwindu Limbangan Kendal khususnya di habitat hutan sekunder, permukiman, DAS dan persawahan. Penelitian dilakukan dengan metode Indeks Point Abudance (IPA atau metode titik hitung.Hasil penelitian ditemukan sebanyak 62 jenis kupu-kupu superfamili Papilionoidae yang terdiri dari 737 individu yang tergolong kedalam empat famili yaitu Papilionidae, Pieridae, Lycaenidae dan Nymphalidae. Hasil analisis indeks keanekaragaman jenis berkisar antara 2,74-3,09, indeks kemerataan jenis berkisar antara 0,86-0,87 dan memiliki dominansi berkisar antara 0,07-0,09. Indeks keanekaragaman jenis dan indeks kemerataan jenis tertinggi tercatat pada habitat permukiman yaitu 3,09 dan 0,87 dan memiliki dominansi 0,07 sedangkan terendah tercatat pada habitat persawahan yaitu 2,74 dan 0,86 dan memiliki dominansi 0,07.Butterfly also contribute in maintaining the ecological balance and enrich biodiversity. The aim of this research was to determine the diversity of butterflies’ superfamily Papilionoidae in Banyuwindu Hamlet Limbangan Sub district Kendal Regency, especially in the secondary forest habitat, settlements, river flow area (RFA and rice field. The population in this research were all kinds of butterflies’ Papilionoidae superfamily in Banyuwindu, Limbangan Kendal. The sample was kind of butterfly superfamily Papilionoidae that observed in Banyuwindu Limbangan Kendal

Functional classification of protein structures by local structure matching in graph representation.

Science.gov (United States)

Mills, Caitlyn L; Garg, Rohan; Lee, Joslynn S; Tian, Liang; Suciu, Alexandru; Cooperman, Gene; Beuning, Penny J; Ondrechen, Mary Jo

2018-03-31

As a result of high-throughput protein structure initiatives, over 14,400 protein structures have been solved by structural genomics (SG) centers and participating research groups. While the totality of SG data represents a tremendous contribution to genomics and structural biology, reliable functional information for these proteins is generally lacking. Better functional predictions for SG proteins will add substantial value to the structural information already obtained. Our method described herein, Graph Representation of Active Sites for Prediction of Function (GRASP-Func), predicts quickly and accurately the biochemical function of proteins by representing residues at the predicted local active site as graphs rather than in Cartesian coordinates. We compare the GRASP-Func method to our previously reported method, structurally aligned local sites of activity (SALSA), using the ribulose phosphate binding barrel (RPBB), 6-hairpin glycosidase (6-HG), and Concanavalin A-like Lectins/Glucanase (CAL/G) superfamilies as test cases. In each of the superfamilies, SALSA and the much faster method GRASP-Func yield similar correct classification of previously characterized proteins, providing a validated benchmark for the new method. In addition, we analyzed SG proteins using our SALSA and GRASP-Func methods to predict function. Forty-one SG proteins in the RPBB superfamily, nine SG proteins in the 6-HG superfamily, and one SG protein in the CAL/G superfamily were successfully classified into one of the functional families in their respective superfamily by both methods. This improved, faster, validated computational method can yield more reliable predictions of function that can be used for a wide variety of applications by the community. © 2018 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

Characterization of the Tetraspan Junctional Complex (4JC) superfamily.

Science.gov (United States)

Chou, Amy; Lee, Andre; Hendargo, Kevin J; Reddy, Vamsee S; Shlykov, Maksim A; Kuppusamykrishnan, Harikrishnan; Medrano-Soto, Arturo; Saier, Milton H

2017-03-01

Connexins or innexins form gap junctions, while claudins and occludins form tight junctions. In this study, statistical data, derived using novel software, indicate that these four junctional protein families and eleven other families of channel and channel auxiliary proteins are related by common descent and comprise the Tetraspan (4 TMS) Junctional Complex (4JC) Superfamily. These proteins all share similar 4 transmembrane α-helical (TMS) topologies. Evidence is presented that they arose via an intragenic duplication event, whereby a 2 TMS-encoding genetic element duplicated tandemly to give 4 TMS proteins. In cases where high resolution structural data were available, the conclusion of homology was supported by conducting structural comparisons. Phylogenetic trees reveal the probable relationships of these 15 families to each other. Long homologues containing fusions to other recognizable domains as well as internally duplicated or fused domains are reported. Large "fusion" proteins containing 4JC domains proved to fall predominantly into family-specific patterns as follows: (1) the 4JC domain was N-terminal; (2) the 4JC domain was C-terminal; (3) the 4JC domain was duplicated or occasionally triplicated and (4) mixed fusion types were present. Our observations provide insight into the evolutionary origins and subfunctions of these proteins as well as guides concerning their structural and functional relationships. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of the S-transferase like superfamily bacillithiol transferases encoded by Bacillus subtilis

Science.gov (United States)

Perera, Varahenage R.; Lapek, John D.; Newton, Gerald L.; Gonzalez, David J.; Pogliano, Kit

2018-01-01

Bacillithiol is a low molecular weight thiol found in Firmicutes that is analogous to glutathione, which is absent in these bacteria. Bacillithiol transferases catalyze the transfer of bacillithiol to various substrates. The S-transferase-like (STL) superfamily contains over 30,000 putative members, including bacillithiol transferases. Proteins in this family are extremely divergent and are related by structural rather than sequence similarity, leaving it unclear if all share the same biochemical activity. Bacillus subtilis encodes eight predicted STL superfamily members, only one of which has been shown to be a bacillithiol transferase. Here we find that the seven remaining proteins show varying levels of metal dependent bacillithiol transferase activity. We have renamed the eight enzymes BstA-H. Mass spectrometry and gene expression studies revealed that all of the enzymes are produced to varying levels during growth and sporulation, with BstB and BstE being the most abundant and BstF and BstH being the least abundant. Interestingly, several bacillithiol transferases are induced in the mother cell during sporulation. A strain lacking all eight bacillithiol transferases showed normal growth in the presence of stressors that adversely affect growth of bacillithiol-deficient strains, such as paraquat and CdCl2. Thus, the STL bacillithiol transferases represent a new group of proteins that play currently unknown, but potentially significant roles in bacillithiol-dependent reactions. We conclude that these enzymes are highly divergent, perhaps to cope with an equally diverse array of endogenous or exogenous toxic metabolites and oxidants. PMID:29451913

Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B)

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Duangtum, Natapol [Medical Molecular Biology Unit, Office for Research and Development Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Junking, Mutita; Sawasdee, Nunghathai [Medical Molecular Biology Unit, Office for Research and Development Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Cheunsuchon, Boonyarit [Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Limjindaporn, Thawornchai, E-mail: limjindaporn@yahoo.com [Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Yenchitsomanus, Pa-thai, E-mail: grpye@mahidol.ac.th [Medical Molecular Biology Unit, Office for Research and Development Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand)

2011-09-16

Highlights: {yields} Impaired trafficking of kAE1 causes distal renal tubular acidosis (dRTA). {yields} The interaction between kAE1 and kinesin family member 3B (KIF3B) is reported. {yields} The co-localization between kAE and KIF3B was detected in human kidney tissues. {yields} A marked reduction of kAE1 on the cell membrane was observed when KIF3B was knockdown. {yields} KFI3B plays an important role in trafficking of kAE1 to the plasma membrane. -- Abstract: Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of {alpha}-intercalated cells of the kidney collecting duct leads to the defect of the Cl{sup -}/HCO{sub 3}{sup -} exchange and the failure of proton (H{sup +}) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney {alpha}-intercalated cells.

Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B)

International Nuclear Information System (INIS)

Duangtum, Natapol; Junking, Mutita; Sawasdee, Nunghathai; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

2011-01-01

Highlights: → Impaired trafficking of kAE1 causes distal renal tubular acidosis (dRTA). → The interaction between kAE1 and kinesin family member 3B (KIF3B) is reported. → The co-localization between kAE and KIF3B was detected in human kidney tissues. → A marked reduction of kAE1 on the cell membrane was observed when KIF3B was knockdown. → KFI3B plays an important role in trafficking of kAE1 to the plasma membrane. -- Abstract: Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of α-intercalated cells of the kidney collecting duct leads to the defect of the Cl - /HCO 3 - exchange and the failure of proton (H + ) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney α-intercalated cells.

Changes in microtubule overlap length regulate kinesin-14-driven microtubule sliding

Czech Academy of Sciences Publication Activity Database

Braun, Marcus; Lánský, Zdeněk; Szuba, A.; Schwarz, F. W.; Mitra, A.; Gao, M.; Luedecke, A.; ten Wolde, P.R.; Diez, S.

2017-01-01

Roč. 13, č. 12 (2017), s. 1245-1252 ISSN 1552-4450 R&D Projects: GA ČR(CZ) GA15-17488S; GA ČR(CZ) GA17-12496Y; GA ČR(CZ) GJ17-12496Y; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : SPINDLE ELONGATION * MITOTIC SPINDLE * KINESIN-5 CIN8 * CROSS-LINKERS Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biophysics Impact factor: 15.066, year: 2016

Evolution of Enzymatic Activities in the Enolase Superfamily: D-Mannonate Dhydratase from Novosphingobium aromaticivorans

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Rakus,J.; Fedorov, A.; Fedorov, E.; Glasner, M.; Vick, J.; Babbitt, P.; Almo, S.; Gerlt, J.

2007-01-01

The d-mannonate dehydratase (ManD) function was assigned to a group of orthologous proteins in the mechanistically diverse enolase superfamily by screening a library of acid sugars. Structures of the wild type ManD from Novosphingobium aromaticivorans were determined at pH 7.5 in the presence of Mg2+ and also in the presence of Mg2+ and the 2-keto-3-keto-d-gluconate dehydration product; the structure of the catalytically active K271E mutant was determined at pH 5.5 in the presence of the d-mannonate substrate. As previously observed in the structures of other members of the enolase superfamily, ManD contains two domains, an N-terminal a+{beta} capping domain and a ({beta}/a)7{beta}-barrel domain. The barrel domain contains the ligands for the essential Mg2+, Asp 210, Glu 236, and Glu 262, at the ends of the third, fourth, and fifth {beta}-strands of the barrel domain, respectively. However, the barrel domain lacks both the Lys acid/base catalyst at the end of the second {beta}-strand and the His-Asp dyad acid/base catalyst at the ends of the seventh and sixth {beta}-strands, respectively, that are found in many members of the superfamily. Instead, a hydrogen-bonded dyad of Tyr 159 in a loop following the second {beta}-strand and Arg 147 at the end of the second {beta}-strand are positioned to initiate the reaction by abstraction of the 2-proton. Both Tyr 159 and His 212, at the end of the third {beta}-strand, are positioned to facilitate both syn-dehydration and ketonization of the resulting enol intermediate to yield the 2-keto-3-keto-d-gluconate product with the observed retention of configuration. The identities and locations of these acid/base catalysts as well as of cationic amino acid residues that stabilize the enolate anion intermediate define a new structural strategy for catalysis (subgroup) in the mechanistically diverse enolase superfamily. With these differences, we provide additional evidence that the ligands for the essential Mg2+ are the only

Genome-wide analysis of the expansin gene superfamily reveals grapevine-specific structural and functional characteristics.

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Silvia Dal Santo

Full Text Available BACKGROUND: Expansins are proteins that loosen plant cell walls in a pH-dependent manner, probably by increasing the relative movement among polymers thus causing irreversible expansion. The expansin superfamily (EXP comprises four distinct families: expansin A (EXPA, expansin B (EXPB, expansin-like A (EXLA and expansin-like B (EXLB. There is experimental evidence that EXPA and EXPB proteins are required for cell expansion and developmental processes involving cell wall modification, whereas the exact functions of EXLA and EXLB remain unclear. The complete grapevine (Vitis vinifera genome sequence has allowed the characterization of many gene families, but an exhaustive genome-wide analysis of expansin gene expression has not been attempted thus far. METHODOLOGY/PRINCIPAL FINDINGS: We identified 29 EXP superfamily genes in the grapevine genome, representing all four EXP families. Members of the same EXP family shared the same exon-intron structure, and phylogenetic analysis confirmed a closer relationship between EXP genes from woody species, i.e. grapevine and poplar (Populus trichocarpa, compared to those from Arabidopsis thaliana and rice (Oryza sativa. We also identified grapevine-specific duplication events involving the EXLB family. Global gene expression analysis confirmed a strong correlation among EXP genes expressed in mature and green/vegetative samples, respectively, as reported for other gene families in the recently-published grapevine gene expression atlas. We also observed the specific co-expression of EXLB genes in woody organs, and the involvement of certain grapevine EXP genes in berry development and post-harvest withering. CONCLUSION: Our comprehensive analysis of the grapevine EXP superfamily confirmed and extended current knowledge about the structural and functional characteristics of this gene family, and also identified properties that are currently unique to grapevine expansin genes. Our data provide a model for the

ATPase Cycle of the Nonmotile Kinesin NOD Allows Microtubule End Tracking and Drives Chromosome Movement

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Cochran, J.; Sindelar, C; Mulko, N; Collins, K; Kong, S; Hawley, R; Kull, F

2009-01-01

Segregation of nonexchange chromosomes during Drosophila melanogaster meiosis requires the proper function of NOD, a nonmotile kinesin-10. We have determined the X-ray crystal structure of the NOD catalytic domain in the ADP- and AMPPNP-bound states. These structures reveal an alternate conformation of the microtubule binding region as well as a nucleotide-sensitive relay of hydrogen bonds at the active site. Additionally, a cryo-electron microscopy reconstruction of the nucleotide-free microtubule-NOD complex shows an atypical binding orientation. Thermodynamic studies show that NOD binds tightly to microtubules in the nucleotide-free state, yet other nucleotide states, including AMPPNP, are weakened. Our pre-steady-state kinetic analysis demonstrates that NOD interaction with microtubules occurs slowly with weak activation of ADP product release. Upon rapid substrate binding, NOD detaches from the microtubule prior to the rate-limiting step of ATP hydrolysis, which is also atypical for a kinesin. We propose a model for NOD's microtubule plus-end tracking that drives chromosome movement.

Dissecting protein loops with a statistical scalpel suggests a functional implication of some structural motifs.

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Regad, Leslie; Martin, Juliette; Camproux, Anne-Claude

2011-06-20

One of the strategies for protein function annotation is to search particular structural motifs that are known to be shared by proteins with a given function. Here, we present a systematic extraction of structural motifs of seven residues from protein loops and we explore their correspondence with functional sites. Our approach is based on the structural alphabet HMM-SA (Hidden Markov Model - Structural Alphabet), which allows simplification of protein structures into uni-dimensional sequences, and advanced pattern statistics adapted to short sequences. Structural motifs of interest are selected by looking for structural motifs significantly over-represented in SCOP superfamilies in protein loops. We discovered two types of structural motifs significantly over-represented in SCOP superfamilies: (i) ubiquitous motifs, shared by several superfamilies and (ii) superfamily-specific motifs, over-represented in few superfamilies. A comparison of ubiquitous words with known small structural motifs shows that they contain well-described motifs as turn, niche or nest motifs. A comparison between superfamily-specific motifs and biological annotations of Swiss-Prot reveals that some of them actually correspond to functional sites involved in the binding sites of small ligands, such as ATP/GTP, NAD(P) and SAH/SAM. Our findings show that statistical over-representation in SCOP superfamilies is linked to functional features. The detection of over-represented motifs within structures simplified by HMM-SA is therefore a promising approach for prediction of functional sites and annotation of uncharacterized proteins.

Dissecting protein loops with a statistical scalpel suggests a functional implication of some structural motifs

Directory of Open Access Journals (Sweden)

Martin Juliette

2011-06-01

Full Text Available Abstract Background One of the strategies for protein function annotation is to search particular structural motifs that are known to be shared by proteins with a given function. Results Here, we present a systematic extraction of structural motifs of seven residues from protein loops and we explore their correspondence with functional sites. Our approach is based on the structural alphabet HMM-SA (Hidden Markov Model - Structural Alphabet, which allows simplification of protein structures into uni-dimensional sequences, and advanced pattern statistics adapted to short sequences. Structural motifs of interest are selected by looking for structural motifs significantly over-represented in SCOP superfamilies in protein loops. We discovered two types of structural motifs significantly over-represented in SCOP superfamilies: (i ubiquitous motifs, shared by several superfamilies and (ii superfamily-specific motifs, over-represented in few superfamilies. A comparison of ubiquitous words with known small structural motifs shows that they contain well-described motifs as turn, niche or nest motifs. A comparison between superfamily-specific motifs and biological annotations of Swiss-Prot reveals that some of them actually correspond to functional sites involved in the binding sites of small ligands, such as ATP/GTP, NAD(P and SAH/SAM. Conclusions Our findings show that statistical over-representation in SCOP superfamilies is linked to functional features. The detection of over-represented motifs within structures simplified by HMM-SA is therefore a promising approach for prediction of functional sites and annotation of uncharacterized proteins.

Superfamily of ankyrin repeat proteins in tomato.

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Yuan, Xiaowei; Zhang, Shizhong; Qing, Xiaohe; Sun, Meihong; Liu, Shiyang; Su, Hongyan; Shu, Huairui; Li, Xinzheng

2013-07-10

The ankyrin repeat (ANK) protein family plays a crucial role in plant growth and development and in response to biotic and abiotic stresses. However, no detailed information concerning this family is available for tomato (Solanum lycopersicum) due to the limited information on whole genome sequences. In this study, we identified a total of 130 ANK genes in tomato genome (SlANK), and these genes were distributed across all 12 chromosomes at various densities. And chromosomal localizations of SlANK genes indicated 25 SlANK genes were involved in tandem duplications. Based on their domain composition, all of the SlANK proteins were grouped into 13 subgroups. A combined phylogenetic tree was constructed with the aligned SlANK protein sequences. This tree revealed that the SlANK proteins comprise five major groups. An analysis of the expression profiles of SlANK genes in tomato in different tissues and in response to stresses showed that the SlANK proteins play roles in plant growth, development and stress responses. To our knowledge, this is the first report of a genome-wide analysis of the tomato ANK gene family. This study provides valuable information regarding the classification and putative functions of SlANK genes in tomato. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Molecular cloning of a peroxisomal Ca2+-dependent member of the mitochondrial carrier superfamily

Science.gov (United States)

Weber, Franz E.; Minestrini, Gianluca; Dyer, James H.; Werder, Moritz; Boffelli, Dario; Compassi, Sabina; Wehrli, Ernst; Thomas, Richard M.; Schulthess, Georg; Hauser, Helmut

1997-01-01

A cDNA from a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA library. The full-length cDNA clone was 3,298 nt long and coded for a protein of 475 amino acids, with four elongation factor-hand motifs located in the N-terminal half of the molecule. The 25-kDa N-terminal polypeptide was expressed in Escherichia coli, and it was demonstrated that it bound Ca2+, undergoing a reversible and specific conformational change as a result. The conformation of the polypeptide was sensitive to Ca2+ which was bound with high affinity (Kd ≈ 0.37 μM), the apparent Hill coefficient for Ca2+-induced changes being about 2.0. The deduced amino acid sequence of the C-terminal half of the molecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP/ATP translocase; this sequence homology identified the protein as a new member of the mitochondrial transporter superfamily. Northern blot analysis revealed the presence of a single transcript of about 3,500 bases, and low expression of the transporter could be detected in the kidney but none in the liver. The main site of expression was the colon with smaller amounts found in the small intestine proximal to the ileum. Immunoelectron microscopy localized the transporter in the peroxisome, although a minor fraction was found in the mitochondria. The Ca2+ binding N-terminal half of the transporter faces the cytosol. PMID:9238007

Engineering of a novel Ca2+-regulated kinesin molecular motor using a calmodulin dimer linker

International Nuclear Information System (INIS)

Shishido, Hideki; Maruta, Shinsaku

2012-01-01

Highlights: ► Engineered kinesin–M13 and calmodulin involving single cysteine were prepared. ► CaM mutant was cross-linked to dimer by bifunctional thiol reactive reagent. ► Kinesin–M13 was dimerized via CaM dimer in the presence of calcium. ► Function of the engineered kinesin was regulated by a Ca 2+ -calmodulin dimer linker. -- Abstract: The kinesin–microtubule system holds great promise as a molecular shuttle device within biochips. However, one current barrier is that such shuttles do not have “on–off” control of their movement. Here we report the development of a novel molecular motor powered by an accelerator and brake system, using a kinesin monomer and a calmodulin (CaM) dimer. The kinesin monomer, K355, was fused with a CaM target peptide (M13 peptide) at the C-terminal part of the neck region (K355–M13). We also prepared CaM dimers using CaM mutants (Q3C), (R86C), or (A147C) and crosslinkers that react with cysteine residues. Following induction of K355–M13 dimerization with CaM dimers, we measured K355–M13 motility and found that it can be reversibly regulated in a Ca 2+ -dependent manner. We also found that velocities of K355–M13 varied depending on the type and crosslink position of the CaM dimer used; crosslink length also had a moderate effect on motility. These results suggest Ca 2+ -dependent dimerization of K355–M13 could be used as a novel molecular shuttle, equipped with an accelerator and brake system, for biochip applications.

Evolutionary history and stress regulation of the lectin superfamily in higher plants

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Ramachandran Srinivasan

2010-03-01

Full Text Available Abstract Background Lectins are a class of carbohydrate-binding proteins. They play roles in various biological processes. However, little is known about their evolutionary history and their functions in plant stress regulation. The availability of full genome sequences from various plant species makes it possible to perform a whole-genome exploration for further understanding their biological functions. Results Higher plant genomes encode large numbers of lectin proteins. Based on their domain structures and phylogenetic analyses, a new classification system has been proposed. In this system, 12 different families have been classified and four of them consist of recently identified plant lectin members. Further analyses show that some of lectin families exhibit species-specific expansion and rapid birth-and-death evolution. Tandem and segmental duplications have been regarded as the major mechanisms to drive lectin expansion although retrogenes also significantly contributed to the birth of new lectin genes in soybean and rice. Evidence shows that lectin genes have been involved in biotic/abiotic stress regulations and tandem/segmental duplications may be regarded as drivers for plants to adapt various environmental stresses through duplication followed by expression divergence. Each member of this gene superfamily may play specialized roles in a specific stress condition and function as a regulator of various environmental factors such as cold, drought and high salinity as well as biotic stresses. Conclusions Our studies provide a new outline of the plant lectin gene superfamily and advance the understanding of plant lectin genes in lineage-specific expansion and their functions in biotic/abiotic stress-related developmental processes.

Anomalous inhibition of c-Met by the kinesin inhibitor aurintricarboxylic acid.

Science.gov (United States)

Milanovic, Mina; Radtke, Simone; Peel, Nick; Howell, Michael; Carrière, Virginie; Joffre, Carine; Kermorgant, Stéphanie; Parker, Peter J

2012-03-01

c-Met [the hepatocyte growth factor (HGF) receptor] is a receptor tyrosine kinase playing a role in various biological events. Overexpression of the receptor has been observed in a number of cancers, correlating with increased metastatic tendency and poor prognosis. Additionally, activating mutations in c-Met kinase domain have been reported in a subset of familial cancers causing resistance to treatment. Receptor trafficking, relying on the integrity of the microtubule network, plays an important role in activation of downstream targets and initiation of signalling events. Aurintricarboxylic acid (ATA) is a triphenylmethane derivative that has been reported to inhibit microtubule motor proteins kinesins. Additional reported properties of this inhibitor include inhibition of protein tyrosine phosphatases, nucleases and members of the Jak family. Here we demonstrate that ATA prevents HGF-induced c-Met phosphorylation, internalisation, subsequent receptor trafficking and degradation. In addition, ATA prevented HGF-induced downstream signalling which also affected cellular function, as assayed by collective cell migration of A549 cells. Surprisingly, the inhibitory effect of ATA on HGF-induced phosphorylation and signalling in vivo was associated with an increase in basal c-Met kinase activity in vitro. It is concluded that the inhibitory effects of ATA on c-Met in vivo is an allosteric effect mediated through the kinase domain of the receptor. As the currently tested adenosine triphosphate competitive tyrosine kinase inhibitors (TKIs) may lead to tumor resistance (McDermott U, et al., Cancer Res 2010;70:1625-34), our findings suggest that novel anti-c-Met therapies could be developed in the future for cancer treatment. Copyright © 2011 UICC.

A Survey of the ATP-Binding Cassette (ABC) Gene Superfamily in the Salmon Louse (Lepeophtheirus salmonis).

Science.gov (United States)

Carmona-Antoñanzas, Greta; Carmichael, Stephen N; Heumann, Jan; Taggart, John B; Gharbi, Karim; Bron, James E; Bekaert, Michaël; Sturm, Armin

2015-01-01

Salmon lice, Lepeophtheirus salmonis (Krøyer, 1837), are fish ectoparasites causing significant economic damage in the mariculture of Atlantic salmon, Salmo salar Linnaeus, 1758. The control of L. salmonis at fish farms relies to a large extent on treatment with anti-parasitic drugs. A problem related to chemical control is the potential for development of resistance, which in L. salmonis is documented for a number of drug classes including organophosphates, pyrethroids and avermectins. The ATP-binding cassette (ABC) gene superfamily is found in all biota and includes a range of drug efflux transporters that can confer drug resistance to cancers and pathogens. Furthermore, some ABC transporters are recognised to be involved in conferral of insecticide resistance. While a number of studies have investigated ABC transporters in L. salmonis, no systematic analysis of the ABC gene family exists for this species. This study presents a genome-wide survey of ABC genes in L. salmonis for which, ABC superfamily members were identified through homology searching of the L. salmonis genome. In addition, ABC proteins were identified in a reference transcriptome of the parasite generated by high-throughput RNA sequencing (RNA-seq) of a multi-stage RNA library. Searches of both genome and transcriptome allowed the identification of a total of 33 genes / transcripts coding for ABC proteins, of which 3 were represented only in the genome and 4 only in the transcriptome. Eighteen sequences were assigned to ABC subfamilies known to contain drug transporters, i.e. subfamilies B (4 sequences), C (11) and G (2). The results suggest that the ABC gene family of L. salmonis possesses fewer members than recorded for other arthropods. The present survey of the L. salmonis ABC gene superfamily will provide the basis for further research into potential roles of ABC transporters in the toxicity of salmon delousing agents and as potential mechanisms of drug resistance.

The structure of hookworm platelet inhibitor (HPI), a CAP superfamily member from Ancylostoma caninum.

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Ma, Dongying; Francischetti, Ivo M B; Ribeiro, Jose M C; Andersen, John F

2015-06-01

Secreted protein components of hookworm species include a number of representatives of the cysteine-rich/antigen 5/pathogenesis-related 1 (CAP) protein family known as Ancylostoma-secreted proteins (ASPs). Some of these have been considered as candidate antigens for the development of vaccines against hookworms. The functions of most CAP superfamily members are poorly understood, but one form, the hookworm platelet inhibitor (HPI), has been isolated as a putative antagonist of the platelet integrins αIIbβ3 and α2β1. Here, the crystal structure of HPI is described and its structural features are examined in relation to its possible function. The HPI structure is similar to those of other ASPs and shows incomplete conservation of the sequence motifs CAP1 and CAP2 that are considered to be diagnostic of CAP superfamily members. The asymmetric unit of the HPI crystal contains a dimer with an extensive interaction interface, but chromatographic measurements indicate that it is primarily monomeric in solution. In the dimeric structure, the putative active-site cleft areas from both monomers are united into a single negatively charged depression. A potential Lys-Gly-Asp disintegrin-like motif was identified in the sequence of HPI, but is not positioned at the apex of a tight turn, making it unlikely that it interacts with the integrin. Recombinant HPI produced in Escherichia coli was found not to inhibit the adhesion of human platelets to collagen or fibrinogen, despite having a native structure as shown by X-ray diffraction. This result corroborates previous analyses of recombinant HPI and suggests that it might require post-translational modification or have a different biological function.

Evolutionary and molecular foundations of multiple contemporary functions of the nitroreductase superfamily.

Science.gov (United States)

Akiva, Eyal; Copp, Janine N; Tokuriki, Nobuhiko; Babbitt, Patricia C

2017-11-07

Insight regarding how diverse enzymatic functions and reactions have evolved from ancestral scaffolds is fundamental to understanding chemical and evolutionary biology, and for the exploitation of enzymes for biotechnology. We undertook an extensive computational analysis using a unique and comprehensive combination of tools that include large-scale phylogenetic reconstruction to determine the sequence, structural, and functional relationships of the functionally diverse flavin mononucleotide-dependent nitroreductase (NTR) superfamily (>24,000 sequences from all domains of life, 54 structures, and >10 enzymatic functions). Our results suggest an evolutionary model in which contemporary subgroups of the superfamily have diverged in a radial manner from a minimal flavin-binding scaffold. We identified the structural design principle for this divergence: Insertions at key positions in the minimal scaffold that, combined with the fixation of key residues, have led to functional specialization. These results will aid future efforts to delineate the emergence of functional diversity in enzyme superfamilies, provide clues for functional inference for superfamily members of unknown function, and facilitate rational redesign of the NTR scaffold. Copyright © 2017 the Author(s). Published by PNAS.

TED, an autonomous and rare maize transposon of the mutator superfamily with a high gametophytic excision frequency.

Science.gov (United States)

Li, Yubin; Harris, Linda; Dooner, Hugo K

2013-09-01

Mutator (Mu) elements, one of the most diverse superfamilies of DNA transposons, are found in all eukaryotic kingdoms, but are particularly numerous in plants. Most of the present knowledge on the transposition behavior of this superfamily comes from studies of the maize (Zea mays) Mu elements, whose transposition is mediated by the autonomous Mutator-Don Robertson (MuDR) element. Here, we describe the maize element TED (for Transposon Ellen Dempsey), an autonomous cousin that differs significantly from MuDR. Element excision and reinsertion appear to require both proteins encoded by MuDR, but only the single protein encoded by TED. Germinal excisions, rare with MuDR, are common with TED, but arise in one of the mitotic divisions of the gametophyte, rather than at meiosis. Instead, transposition-deficient elements arise at meiosis, suggesting that the double-strand breaks produced by element excision are repaired differently in mitosis and meiosis. Unlike MuDR, TED is a very low-copy transposon whose number and activity do not undergo dramatic changes upon inbreeding or outcrossing. Like MuDR, TED transposes mostly to unlinked sites and can form circular transposition products. Sequences closer to TED than to MuDR were detected only in the grasses, suggesting a rather recent evolutionary split from a common ancestor.

Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey.

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Aggarwal, Bharat B; Gupta, Subash C; Kim, Ji Hye

2012-01-19

Although activity that induced tumor regression was observed and termed tumor necrosis factor (TNF) as early as the 1960s, the true identity of TNF was not clear until 1984, when Aggarwal and coworkers reported, for the first time, the isolation of 2 cytotoxic factors: one, derived from macrophages (molecular mass 17 kDa), was named TNF, and the second, derived from lymphocytes (20 kDa), was named lymphotoxin. Because the 2 cytotoxic factors exhibited 50% amino acid sequence homology and bound to the same receptor, they came to be called TNF-α and TNF-β. Identification of the protein sequences led to cloning of their cDNA. Based on sequence homology to TNF-α, now a total of 19 members of the TNF superfamily have been identified, along with 29 interacting receptors, and several molecules that interact with the cytoplasmic domain of these receptors. The roles of the TNF superfamily in inflammation, apoptosis, proliferation, invasion, angiogenesis, metastasis, and morphogenesis have been documented. Their roles in immunologic, cardiovascular, neurologic, pulmonary, and metabolic diseases are becoming apparent. TNF superfamily members are active targets for drug development, as indicated by the recent approval and expanding market of TNF blockers used to treat rheumatoid arthritis, psoriasis, Crohns disease, and osteoporosis, with a total market of more than US $20 billion. As we learn more about this family, more therapeutics will probably emerge. In this review, we summarize the initial discovery of TNF-α, and the insights gained regarding the roles of this molecule and its related family members in normal physiology and disease.

Motif III in superfamily 2 "helicases" helps convert the binding energy of ATP into a high-affinity RNA binding site in the yeast DEAD-box protein Ded1.

Science.gov (United States)

Banroques, Josette; Doère, Monique; Dreyfus, Marc; Linder, Patrick; Tanner, N Kyle

2010-03-05

Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP. (c) 2009 Elsevier Ltd. All rights reserved.

Diffusion as a Ruler: Modeling Kinesin Diffusion as a Length Sensor for Intraflagellar Transport.

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Hendel, Nathan L; Thomson, Matthew; Marshall, Wallace F

2018-02-06

An important question in cell biology is whether cells are able to measure size, either whole cell size or organelle size. Perhaps cells have an internal chemical representation of size that can be used to precisely regulate growth, or perhaps size is just an accident that emerges due to constraint of nutrients. The eukaryotic flagellum is an ideal model for studying size sensing and control because its linear geometry makes it essentially one-dimensional, greatly simplifying mathematical modeling. The assembly of flagella is regulated by intraflagellar transport (IFT), in which kinesin motors carry cargo adaptors for flagellar proteins along the flagellum and then deposit them at the tip, lengthening the flagellum. The rate at which IFT motors are recruited to begin transport into the flagellum is anticorrelated with the flagellar length, implying some kind of communication between the base and the tip and possibly indicating that cells contain some mechanism for measuring flagellar length. Although it is possible to imagine many complex scenarios in which additional signaling molecules sense length and carry feedback signals to the cell body to control IFT, might the already-known components of the IFT system be sufficient to allow length dependence of IFT? Here we investigate a model in which the anterograde kinesin motors unbind after cargo delivery, diffuse back to the base, and are subsequently reused to power entry of new IFT trains into the flagellum. By mathematically modeling and simulating such a system, we are able to show that the diffusion time of the motors can in principle be sufficient to serve as a proxy for length measurement. We found that the diffusion model can not only achieve a stable steady-state length without the addition of any other signaling molecules or pathways, but also is able to produce the anticorrelation between length and IFT recruitment rate that has been observed in quantitative imaging studies. Copyright © 2017 Biophysical

Transient receptor potential channel superfamily: Role in lower urinary tract function.

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Ogawa, Teruyuki; Imamura, Tetsuya; Nakazawa, Masaki; Hiragata, Shiro; Nagai, Takashi; Minagawa, Tomonori; Yokoyama, Hitoshi; Ishikawa, Masakuni; Domen, Takahisa; Ishizuka, Osamu

2015-11-01

Lower urinary tract symptoms associated with neurogenic bladder and overactive bladder syndrome are mediated in part by members of the transient receptor potential channel superfamily. The best studied member of this superfamily is the vanilloid receptor. Other transient receptor potential channels, such as the melastatin receptor and the ankyrin receptor, are also active in the pathogenesis of lower urinary tract dysfunction. However, the detailed mechanisms by which the transient receptor potential channels contribute to lower urinary tract symptoms are still not clear, and the therapeutic benefits of modulating transient receptor potential channel activity have not been proved in the clinical setting. In the present review, to better understand the pathophysiology and therapeutic potential for lower urinary tract symptoms, we summarize the presence and role of different members of the transient receptor potential channel superfamily in the lower urinary tract. © 2015 The Japanese Urological Association.

Homology models guide discovery of diverse enzyme specificities among dipeptide epimerases in the enolase superfamily

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Lukk, Tiit; Sakai, Ayano; Kalyanaraman, Chakrapani; Brown, Shoshana D.; Imker, Heidi J.; Song, Ling; Fedorov, Alexander A.; Fedorov, Elena V.; Toro, Rafael; Hillerich, Brandan; Seidel, Ronald; Patskovsky, Yury; Vetting, Matthew W.; Nair, Satish K.; Babbitt, Patricia C.; Almo, Steven C.; Gerlt, John A.; Jacobson, Matthew P.

2012-01-01

The rapid advance in genome sequencing presents substantial challenges for protein functional assignment, with half or more of new protein sequences inferred from these genomes having uncertain assignments. The assignment of enzyme function in functionally diverse superfamilies represents a particular challenge, which we address through a combination of computational predictions, enzymology, and structural biology. Here we describe the results of a focused investigation of a group of enzymes in the enolase superfamily that are involved in epimerizing dipeptides. The first members of this group to be functionally characterized were Ala-Glu epimerases in Eschericiha coli and Bacillus subtilis, based on the operon context and enzymological studies; these enzymes are presumed to be involved in peptidoglycan recycling. We have subsequently studied more than 65 related enzymes by computational methods, including homology modeling and metabolite docking, which suggested that many would have divergent specificities;, i.e., they are likely to have different (unknown) biological roles. In addition to the Ala-Phe epimerase specificity reported previously, we describe the prediction and experimental verification of: (i) a new group of presumed Ala-Glu epimerases; (ii) several enzymes with specificity for hydrophobic dipeptides, including one from Cytophaga hutchinsonii that epimerizes D-Ala-D-Ala; and (iii) a small group of enzymes that epimerize cationic dipeptides. Crystal structures for certain of these enzymes further elucidate the structural basis of the specificities. The results highlight the potential of computational methods to guide experimental characterization of enzymes in an automated, large-scale fashion. PMID:22392983

Improving Protein Fold Recognition by Deep Learning Networks

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Jo, Taeho; Hou, Jie; Eickholt, Jesse; Cheng, Jianlin

2015-12-01

For accurate recognition of protein folds, a deep learning network method (DN-Fold) was developed to predict if a given query-template protein pair belongs to the same structural fold. The input used stemmed from the protein sequence and structural features extracted from the protein pair. We evaluated the performance of DN-Fold along with 18 different methods on Lindahl’s benchmark dataset and on a large benchmark set extracted from SCOP 1.75 consisting of about one million protein pairs, at three different levels of fold recognition (i.e., protein family, superfamily, and fold) depending on the evolutionary distance between protein sequences. The correct recognition rate of ensembled DN-Fold for Top 1 predictions is 84.5%, 61.5%, and 33.6% and for Top 5 is 91.2%, 76.5%, and 60.7% at family, superfamily, and fold levels, respectively. We also evaluated the performance of single DN-Fold (DN-FoldS), which showed the comparable results at the level of family and superfamily, compared to ensemble DN-Fold. Finally, we extended the binary classification problem of fold recognition to real-value regression task, which also show a promising performance. DN-Fold is freely available through a web server at http://iris.rnet.missouri.edu/dnfold.

Improving Protein Fold Recognition by Deep Learning Networks.

Science.gov (United States)

Jo, Taeho; Hou, Jie; Eickholt, Jesse; Cheng, Jianlin

2015-12-04

For accurate recognition of protein folds, a deep learning network method (DN-Fold) was developed to predict if a given query-template protein pair belongs to the same structural fold. The input used stemmed from the protein sequence and structural features extracted from the protein pair. We evaluated the performance of DN-Fold along with 18 different methods on Lindahl's benchmark dataset and on a large benchmark set extracted from SCOP 1.75 consisting of about one million protein pairs, at three different levels of fold recognition (i.e., protein family, superfamily, and fold) depending on the evolutionary distance between protein sequences. The correct recognition rate of ensembled DN-Fold for Top 1 predictions is 84.5%, 61.5%, and 33.6% and for Top 5 is 91.2%, 76.5%, and 60.7% at family, superfamily, and fold levels, respectively. We also evaluated the performance of single DN-Fold (DN-FoldS), which showed the comparable results at the level of family and superfamily, compared to ensemble DN-Fold. Finally, we extended the binary classification problem of fold recognition to real-value regression task, which also show a promising performance. DN-Fold is freely available through a web server at http://iris.rnet.missouri.edu/dnfold.

TED, an Autonomous and Rare Maize Transposon of the Mutator Superfamily with a High Gametophytic Excision Frequency[W

Science.gov (United States)

Li, Yubin; Harris, Linda; Dooner, Hugo K.

2013-01-01

Mutator (Mu) elements, one of the most diverse superfamilies of DNA transposons, are found in all eukaryotic kingdoms, but are particularly numerous in plants. Most of the present knowledge on the transposition behavior of this superfamily comes from studies of the maize (Zea mays) Mu elements, whose transposition is mediated by the autonomous Mutator-Don Robertson (MuDR) element. Here, we describe the maize element TED (for Transposon Ellen Dempsey), an autonomous cousin that differs significantly from MuDR. Element excision and reinsertion appear to require both proteins encoded by MuDR, but only the single protein encoded by TED. Germinal excisions, rare with MuDR, are common with TED, but arise in one of the mitotic divisions of the gametophyte, rather than at meiosis. Instead, transposition-deficient elements arise at meiosis, suggesting that the double-strand breaks produced by element excision are repaired differently in mitosis and meiosis. Unlike MuDR, TED is a very low-copy transposon whose number and activity do not undergo dramatic changes upon inbreeding or outcrossing. Like MuDR, TED transposes mostly to unlinked sites and can form circular transposition products. Sequences closer to TED than to MuDR were detected only in the grasses, suggesting a rather recent evolutionary split from a common ancestor. PMID:24038653

Diversification of a single ancestral gene into a successful toxin superfamily in highly venomous Australian funnel-web spiders.

Science.gov (United States)

Pineda, Sandy S; Sollod, Brianna L; Wilson, David; Darling, Aaron; Sunagar, Kartik; Undheim, Eivind A B; Kely, Laurence; Antunes, Agostinho; Fry, Bryan G; King, Glenn F

2014-03-05

Spiders have evolved pharmacologically complex venoms that serve to rapidly subdue prey and deter predators. The major toxic factors in most spider venoms are small, disulfide-rich peptides. While there is abundant evidence that snake venoms evolved by recruitment of genes encoding normal body proteins followed by extensive gene duplication accompanied by explosive structural and functional diversification, the evolutionary trajectory of spider-venom peptides is less clear. Here we present evidence of a spider-toxin superfamily encoding a high degree of sequence and functional diversity that has evolved via accelerated duplication and diversification of a single ancestral gene. The peptides within this toxin superfamily are translated as prepropeptides that are posttranslationally processed to yield the mature toxin. The N-terminal signal sequence, as well as the protease recognition site at the junction of the propeptide and mature toxin are conserved, whereas the remainder of the propeptide and mature toxin sequences are variable. All toxin transcripts within this superfamily exhibit a striking cysteine codon bias. We show that different pharmacological classes of toxins within this peptide superfamily evolved under different evolutionary selection pressures. Overall, this study reinforces the hypothesis that spiders use a combinatorial peptide library strategy to evolve a complex cocktail of peptide toxins that target neuronal receptors and ion channels in prey and predators. We show that the ω-hexatoxins that target insect voltage-gated calcium channels evolved under the influence of positive Darwinian selection in an episodic fashion, whereas the κ-hexatoxins that target insect calcium-activated potassium channels appear to be under negative selection. A majority of the diversifying sites in the ω-hexatoxins are concentrated on the molecular surface of the toxins, thereby facilitating neofunctionalisation leading to new toxin pharmacology.

A smart dust biosensor powered by kinesin motors.

Science.gov (United States)

Fischer, Thorsten; Agarwal, Ashutosh; Hess, Henry

2009-03-01

Biosensors can be miniaturized by either injecting smaller volumes into micro- and nanofluidic devices or immersing increasingly sophisticated particles known as 'smart dust' into the sample. The term 'smart dust' originally referred to cubic-millimetre wireless semiconducting sensor devices that could invisibly monitor the environment in buildings and public spaces, but later it also came to include functional micrometre-sized porous silicon particles used to monitor yet smaller environments. The principal challenge in designing smart dust biosensors is integrating transport functions with energy supply into the device. Here, we report a hybrid microdevice that is powered by ATP and relies on antibody-functionalized microtubules and kinesin motors to transport the target analyte into a detection region. The transport step replaces the wash step in traditional double-antibody sandwich assays. Owing to their small size and autonomous function, we envision that large numbers of such smart dust biosensors could be inserted into organisms or distributed into the environment for remote sensing.

Structural and Biochemical Investigation of PglF from Campylobacter jejuni Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily

Energy Technology Data Exchange (ETDEWEB)

Riegert, Alexander S. [Department; Thoden, James B. [Department; Schoenhofen, Ian C. [National; Watson, David C. [National; Young, N. Martin [National; Tipton, Peter A. [Department; Holden, Hazel M. [Department

2017-11-03

Within recent years it has become apparent that protein glycosylation is not limited to eukaryotes. Indeed, in Campylobacter jejuni, a Gram-negative bacterium, more than 60 of its proteins are known to be glycosylated. One of the sugars found in such glycosylated proteins is 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, hereafter referred to as QuiNAc4NAc. The pathway for its biosynthesis, initiating with UDP-GlcNAc, requires three enzymes referred to as PglF, PglE, and PlgD. The focus of this investigation is on PglF, an NAD+-dependent sugar 4,6-dehydratase known to belong to the short chain dehydrogenase/reductase (SDR) superfamily. Specifically, PglF catalyzes the first step in the pathway, namely, the dehydration of UDP-GlcNAc to UDP-2-acetamido-2,6-dideoxy-α-d-xylo-hexos-4-ulose. Most members of the SDR superfamily contain a characteristic signature sequence of YXXXK where the conserved tyrosine functions as a catalytic acid or a base. Strikingly, in PglF, this residue is a methionine. Here we describe a detailed structural and functional investigation of PglF from C. jejuni. For this investigation five X-ray structures were determined to resolutions of 2.0 Å or better. In addition, kinetic analyses of the wild-type and site-directed variants were performed. On the basis of the data reported herein, a new catalytic mechanism for a SDR superfamily member is proposed that does not require the typically conserved tyrosine residue.

Structural analysis of papain-like NlpC/P60 superfamily enzymes with a circularly permuted topology reveals potential lipid binding sites.

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Qingping Xu

Full Text Available NlpC/P60 superfamily papain-like enzymes play important roles in all kingdoms of life. Two members of this superfamily, LRAT-like and YaeF/YiiX-like families, were predicted to contain a catalytic domain that is circularly permuted such that the catalytic cysteine is located near the C-terminus, instead of at the N-terminus. These permuted enzymes are widespread in virus, pathogenic bacteria, and eukaryotes. We determined the crystal structure of a member of the YaeF/YiiX-like family from Bacillus cereus in complex with lysine. The structure, which adopts a ligand-induced, "closed" conformation, confirms the circular permutation of catalytic residues. A comparative analysis of other related protein structures within the NlpC/P60 superfamily is presented. Permutated NlpC/P60 enzymes contain a similar conserved core and arrangement of catalytic residues, including a Cys/His-containing triad and an additional conserved tyrosine. More surprisingly, permuted enzymes have a hydrophobic S1 binding pocket that is distinct from previously characterized enzymes in the family, indicative of novel substrate specificity. Further analysis of a structural homolog, YiiX (PDB 2if6 identified a fatty acid in the conserved hydrophobic pocket, thus providing additional insights into possible function of these novel enzymes.

Crystal structure and potential physiological role of zebra fish thioesterase superfamily member 2 (fTHEM2)

Energy Technology Data Exchange (ETDEWEB)

Yu, Shanshan; Li, Han; Gao, Feng; Zhou, Ying, E-mail: zhouying@moon.ibp.ac.cn

2015-08-07

Thioesterase superfamily member 2 (THEM2) is an essential protein for mammalian cell proliferation. It belongs to the hotdog-fold thioesterase superfamily and catalyzes hydrolysis of thioester bonds of acyl-CoA in vitro, while its in vivo function remains unrevealed. In this study, Zebra fish was selected as a model organism to facilitate the investigations on THEM2. First, we solved the crystal structure of recombinant fTHEM2 at the resolution of 1.80 Å, which displayed a similar scaffolding as hTHEM2. Second, functional studies demonstrated that fTHEM2 is capable of hydrolyzing palmitoyl-CoA in vitro. In addition, injection of morpholino against fTHEM2 at one-cell stage resulted in distorted early embryo development, including delayed cell division, retarded development and increased death rate. The above findings validated our hypothesis that fTHEM2 could serve as an ideal surrogate for studying the physiological functions of THEM2. - Highlights: • The crystal structure of recombinant fTHEM2 is presented. • fTHEM2 is capable of hydrolyzing palmitoyl-CoA. • The influence of fTHEM2 on early embryo development is demonstrated.

Identification, immunolocalization, and characterization analyses of an exopeptidase of papain superfamily, (cathepsin C) from Clonorchis sinensis.

Science.gov (United States)

Liang, Pei; He, Lei; Xu, Yanquan; Chen, Xueqing; Huang, Yan; Ren, Mengyu; Liang, Chi; Li, Xuerong; Xu, Jin; Lu, Gang; Yu, Xinbing

2014-10-01

Cathepsin C is an important exopeptidase of papain superfamily and plays a number of great important roles during the parasitic life cycle. The amino acid sequence of cathepsin C from Clonorchis sinensis (C. sinensis) showed 54, 53, and 49% identities to that of Schistosoma japonicum, Schistosoma mansoni, and Homo sapiens, respectively. Phylogenetic analysis utilizing the sequences of papain superfamily of C. sinensis demonstrated that cathepsin C and cathepsin Bs came from a common ancestry. Cathepsin C of C. sinensis (Cscathepsin C) was identified as an excretory/secretory product by Western blot analysis. The results of transcriptional level and translational level of Cscathepsin C at metacercaria stage were higher than that at adult worms. Immunolocalization analysis indicated that Cscathepsin C was specifically distributed in the suckers (oral sucker and ventral sucker), eggs, vitellarium, intestines, and testis of adult worms. In the metacercaria, it was mainly detected on the cyst wall and excretory bladder. Combining with the results mentioned above, it implies that Cscathepsin C may be an essential proteolytic enzyme for proteins digestion of hosts, nutrition assimilation, and immune invasion of C. sinensis. Furthermore, it may be a potential diagnostic antigen and drug target against C. sinensis infection.

Experimental protocols for and studies of the effects of surface passivation and water isotopes on the gliding speed of microtubules propelled by kinesin-1

Science.gov (United States)

Maloney, Roger Andrew

This dissertation explores how the kinesin-1 and microtubule system is affected by surface passivation and water isotopes. Surface passivation was found to affect the gliding speed that microtubules exhibit in the gliding motility assay and the lengths of microtubules supported by the passivation. It was also found that gliding speeds of microtubules are very sensitive to temperature changes. Studies changing the water isotope were a first attempt to investigate if changing the solvent changed the osmotic pressure of the solution kinesin and microtubules were in. No osmotic pressure changes were observed, however, the experiments using different isotopes of water did illuminate the possibility that kinesin may be sensitive to viscosity changes in the solvent. This experiment also suggests further experiments that can be specifically designed to probe osmotic pressure changes. This thesis was also the first thesis ever, to the best of the author's knowledge, to be done in a completely open format. All information and notebook entries that are related to it, as well as the thesis itself, can be found on the website OpenWetWare. The thesis can also be found there including all the different versions that went into its editing. The philosophy and process of making data open and accessible to every one is also discussed.

Small-angle X-ray scattering analysis reveals the ATP-bound monomeric state of the ATPase domain from the homodimeric MutL endonuclease, a GHKL phosphotransferase superfamily protein.

Science.gov (United States)

Iino, Hitoshi; Hikima, Takaaki; Nishida, Yuya; Yamamoto, Masaki; Kuramitsu, Seiki; Fukui, Kenji

2015-05-01

DNA mismatch repair is an excision system that removes mismatched bases chiefly generated by replication errors. In this system, MutL endonucleases direct the excision reaction to the error-containing strand of the duplex by specifically incising the newly synthesized strand. Both bacterial homodimeric and eukaryotic heterodimeric MutL proteins belong to the GHKL ATPase/kinase superfamily that comprises the N-terminal ATPase and C-terminal dimerization regions. Generally, the GHKL proteins show large ATPase cycle-dependent conformational changes, including dimerization-coupled ATP binding of the N-terminal domain. Interestingly, the ATPase domain of human PMS2, a subunit of the MutL heterodimer, binds ATP without dimerization. The monomeric ATP-bound state of the domain has been thought to be characteristic of heterodimeric GHKL proteins. In this study, we characterized the ATP-bound state of the ATPase domain from the Aquifex aeolicus MutL endonuclease, which is a homodimeric GHKL protein unlike the eukaryotic MutL. Gel filtration, dynamic light scattering, and small-angle X-ray scattering analyses clearly showed that the domain binds ATP in a monomeric form despite its homodimeric nature. This indicates that the uncoupling of dimerization and ATP binding is a common feature among bacterial and eukaryotic MutL endonucleases, which we suggest is closely related to the molecular mechanisms underlying mismatch repair.

A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily.

Science.gov (United States)

Sang, Pau Biak; Srinath, Thiruneelakantan; Patil, Aravind Goud; Woo, Eui-Jeon; Varshney, Umesh

2015-09-30

Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

New O-superfamily conotoxins from Conus striatus inhabited near Chinese Hainan Island

Institute of Scientific and Technical Information of China (English)

无

2000-01-01

Conotoxins are short peptide-toxins with specific targets and large diversity.They are useful in analgesia,neuroprotection,detection of some kinds of deseases,and receptor and ion channel study.In order to explore the conotoxin resourses of Chinese oceans,rapid amplification of 3' cDNA ends (RACE) method was utilized to systemically analyze the O-superfamily conotoxin content of Conus striatus inhabited near Chinese Hainan Island.Six new O-superfamily conopeptides were identified,one of which is highly homologous to MVIIA,an N-type calcium channel antagonist.

Bacterial Multidrug Efflux Pumps of the Major Facilitator Superfamily as Targets for Modulation.

Science.gov (United States)

Kumar, Sanath; He, Guixin; Kakarla, Prathusha; Shrestha, Ugina; Ranjana, K C; Ranaweera, Indrika; Willmon, T Mark; Barr, Sharla R; Hernandez, Alberto J; Varela, Manuel F

2016-01-01

Causative agents of infectious disease that are multidrug resistant bacterial pathogens represent a serious public health concern due to the increasingly difficult nature of achieving efficacious clinical treatments. Of the various acquired and intrinsic antimicrobial agent resistance determinants, integral-membrane multidrug efflux pumps of the major facilitator superfamily constitute a major mechanism of bacterial resistance. The major facilitator superfamily (MFS) encompasses thousands of known related secondary active and passive solute transporters, including multidrug efflux pumps, from bacteria to humans. This review article addresses recent developments involving the targeting by various modulators of bacterial multidrug efflux pumps from the major facilitator superfamily. It is currently of tremendous interest to modulate bacterial multidrug efflux pumps in order to eventually restore the clinical efficacy of therapeutic agents against recalcitrant bacterial infections. Such MFS multidrug efflux pumps are good targets for modulation.

A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria

Science.gov (United States)

Quiles-Puchalt, Nuria; Tormo-Más, María Ángeles; Campoy, Susana; Toledo-Arana, Alejandro; Monedero, Vicente; Lasa, Íñigo; Novick, Richard P.; Christie, Gail E.; Penadés, José R.

2013-01-01

The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria. PMID:23771138

The Anabaena sensory rhodopsin transducer defines a novel superfamily of prokaryotic small-molecule binding domains

Directory of Open Access Journals (Sweden)

De Souza Robson F

2009-08-01

Full Text Available Abstract The Anabaena sensory rhodopsin transducer (ASRT is a small protein that has been claimed to function as a signaling molecule downstream of the cyanobacterial sensory rhodopsin. However, orthologs of ASRT have been detected in several bacteria that lack rhodopsin, raising questions about the generality of this function. Using sequence profile searches we show that ASRT defines a novel superfamily of β-sandwich fold domains. Through contextual inference based on domain architectures and predicted operons and structural analysis we present strong evidence that these domains bind small molecules, most probably sugars. We propose that the intracellular versions like ASRT probably participate as sensors that regulate a diverse range of sugar metabolism operons or even the light sensory behavior in Anabaena by binding sugars or related metabolites. We also show that one of the extracellular versions define a predicted sugar-binding structure in a novel cell-surface lipoprotein found across actinobacteria, including several pathogens such as Tropheryma, Actinomyces and Thermobifida. The analysis of this superfamily also provides new data to investigate the evolution of carbohydrate binding modes in β-sandwich domains with very different topologies. Reviewers: This article was reviewed by M. Madan Babu and Mark A. Ragan.

Probing intracellular motor protein activity using an inducible cargo trafficking assay.

Science.gov (United States)

Kapitein, Lukas C; Schlager, Max A; van der Zwan, Wouter A; Wulf, Phebe S; Keijzer, Nanda; Hoogenraad, Casper C

2010-10-06

Although purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living cells, we utilized the FKBP-rapalog-FRB heterodimerization system to develop an in vivo peroxisomal trafficking assay that allows inducible recruitment of exogenous and endogenous kinesin, dynein, and myosin motors to drive specific cargo transport. We demonstrate that cargo rapidly redistributes with distinct dynamics for each respective motor, and that combined (antagonistic) actions of more complex motor combinations can also be probed. Of importance, robust cargo redistribution is readily achieved by one type of motor protein and does not require the presence of opposite-polarity motors. Simultaneous live-cell imaging of microtubules and kinesin or dynein-propelled peroxisomes, combined with high-resolution particle tracking, revealed that peroxisomes frequently pause at microtubule intersections. Titration and washout experiments furthermore revealed that motor recruitment by rapalog-induced heterodimerization is dose-dependent but irreversible. Our assay directly demonstrates that robust cargo motility does not require the presence of opposite-polarity motors, and can therefore be used to characterize the motile properties of specific types of motor proteins. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Structure of the d-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes

International Nuclear Information System (INIS)

Bera, Asim K.; Atanasova, Vesna; Gamage, Swarna; Robinson, Howard; Parsons, James F.

2010-01-01

The structure of EhpF from P. agglomerans has been solved alone and in complex with phenazine-1,6-dicarboxylate. Apo EhpF was solved and refined in two different space groups at 1.95 and 2.3 Å resolution and the EhpF–phenazine-1,6-dicarboxylate complex structure was determined at 2.8 Å resolution. The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound d-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate

Nuclear Receptors in atherosclerosis: a superfamily with many 'Goodfellas'

NARCIS (Netherlands)

Kurakula, Kondababu; Hamers, Anouk A. J.; de Waard, Vivian; de Vries, Carlie J. M.

2013-01-01

Nuclear Receptors form a superfamily of 48 transcription factors that exhibit a plethora of functions in steroid hormone signaling, regulation of metabolism, circadian rhythm and cellular differentiation. In this review, we describe our current knowledge on the role of Nuclear Receptors in

Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

Science.gov (United States)

Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

Relationship between Apolipoprotein Superfamily and Parkinson's Disease

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Lin Li

2017-01-01

Conclusions: The Apo superfamily has been proved to be closely involved in the initiation, progression, and prognosis of PD. Apos and their genes are of great value in predicting the susceptibility of PD and hopeful to become the target of medical intervention to prevent the onset of PD or slow down the progress. Therefore, further large-scale studies are warranted to elucidate the precise mechanisms of Apos in PD.

D-Ribulose 5-Phosphate 3-Epimerase: Functional and Structural Relationships to Members of the Ribulose-Phosphate Binding (beta/alpha)8-Barrel Superfamily

International Nuclear Information System (INIS)

Akana, J.; Federov, A.; Federov, E.; Novak, W.; Babbitt, P.; Almo, S.; Gerlt, J.

2006-01-01

The 'ribulose phosphate binding' superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (β/α) 8 -barrel ancestor. The superfamily includes D-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice and Plasmodium falciparum, the RPE from Streptococcus pyogenes is activated by Zn 2+ which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn 2+ and inactive apoenzyme cannot be prepared, the affinity for Zn 2+ is decreased by alanine substitutions for the two histidine residues that coordinate the Zn 2+ ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn 2+ . The crystal structure of the RPE was solved at 1.8 Angstroms resolution in the presence of D-xylitol 5-phosphate, an inert analogue of the D-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn 2+ that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn 2+ and participate as acid/base catalysts are not conserved. We conclude that only the phosphate

D-Ribulose 5-Phosphate 3-Epimerase: Functional and Structural Relationships to Members of the Ribulose-Phosphate Binding (beta/alpha)8-Barrel Superfamily

Energy Technology Data Exchange (ETDEWEB)

Akana,J.; Federov, A.; Federov, E.; Novak, W.; Babbitt, P.; Almo, S.; Gerlt, J.

2006-01-01

The 'ribulose phosphate binding' superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common ({beta}/{alpha}){sub 8}-barrel ancestor. The superfamily includes D-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice and Plasmodium falciparum, the RPE from Streptococcus pyogenes is activated by Zn{sup 2+} which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn{sup 2+} and inactive apoenzyme cannot be prepared, the affinity for Zn{sup 2+} is decreased by alanine substitutions for the two histidine residues that coordinate the Zn{sup 2+} ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn{sup 2+}. The crystal structure of the RPE was solved at 1.8 Angstroms resolution in the presence of D-xylitol 5-phosphate, an inert analogue of the D-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn{sup 2+} that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn{sup 2+} and participate as acid/base catalysts

Different functional modes of BAR domain proteins in formation and plasticity of mammalian postsynapses.

Science.gov (United States)

Kessels, Michael M; Qualmann, Britta

2015-09-01

A plethora of cell biological processes involve modulations of cellular membranes. By using extended lipid-binding interfaces, some proteins have the power to shape membranes by attaching to them. Among such membrane shapers, the superfamily of Bin-Amphiphysin-Rvs (BAR) domain proteins has recently taken center stage. Extensive structural work on BAR domains has revealed a common curved fold that can serve as an extended membrane-binding interface to modulate membrane topologies and has allowed the grouping of the BAR domain superfamily into subfamilies with structurally slightly distinct BAR domain subtypes (N-BAR, BAR, F-BAR and I-BAR). Most BAR superfamily members are expressed in the mammalian nervous system. Neurons are elaborately shaped and highly compartmentalized cells. Therefore, analyses of synapse formation and of postsynaptic reorganization processes (synaptic plasticity) - a basis for learning and memory formation - has unveiled important physiological functions of BAR domain superfamily members. These recent advances, furthermore, have revealed that the functions of BAR domain proteins include different aspects. These functions are influenced by the often complex domain organization of BAR domain proteins. In this Commentary, we review these recent insights and propose to classify BAR domain protein functions into (1) membrane shaping, (2) physical integration, (3) action through signaling components, and (4) suppression of other BAR domain functions. © 2015. Published by The Company of Biologists Ltd.

The structure and function of endophilin proteins

DEFF Research Database (Denmark)

Kjaerulff, Ole; Brodin, Lennart; Jung, Anita

2011-01-01

Members of the BAR domain protein superfamily are essential elements of cellular traffic. Endophilins are among the best studied BAR domain proteins. They have a prominent function in synaptic vesicle endocytosis (SVE), receptor trafficking and apoptosis, and in other processes that require...

The Kinesin Adaptor Calsyntenin-1 Organizes Microtubule Polarity and Regulates Dynamics during Sensory Axon Arbor Development

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Mary C. Halloran

2017-04-01

Full Text Available Axon growth and branching, and development of neuronal polarity are critically dependent on proper organization and dynamics of the microtubule (MT cytoskeleton. MTs must organize with correct polarity for delivery of diverse cargos to appropriate subcellular locations, yet the molecular mechanisms regulating MT polarity remain poorly understood. Moreover, how an actively branching axon reorganizes MTs to direct their plus ends distally at branch points is unknown. We used high-speed, in vivo imaging of polymerizing MT plus ends to characterize MT dynamics in developing sensory axon arbors in zebrafish embryos. We find that axonal MTs are highly dynamic throughout development, and that the peripheral and central axons of sensory neurons show differences in MT behaviors. Furthermore, we show that Calsyntenin-1 (Clstn-1, a kinesin adaptor required for sensory axon branching, also regulates MT polarity in developing axon arbors. In wild type neurons the vast majority of MTs are directed in the correct plus-end-distal orientation from early stages of development. Loss of Clstn-1 causes an increase in MTs polymerizing in the retrograde direction. These misoriented MTs most often are found near growth cones and branch points, suggesting Clstn-1 is particularly important for organizing MT polarity at these locations. Together, our results suggest that Clstn-1, in addition to regulating kinesin-mediated cargo transport, also organizes the underlying MT highway during axon arbor development.

Diversity of function in the isocitrate lyase enzyme superfamily: the Dianthus caryophyllus petal death protein cleaves alpha-keto and alpha-hydroxycarboxylic acids.

Science.gov (United States)

Lu, Zhibing; Feng, Xiaohua; Song, Ling; Han, Ying; Kim, Alexander; Herzberg, Osnat; Woodson, William R; Martin, Brian M; Mariano, Patrick S; Dunaway-Mariano, Debra

2005-12-20

The work described in this paper was carried out to define the chemical function a new member of the isocitrate lyase enzyme family derived from the flowering plant Dianthus caryophyllus. This protein (Swiss-Prot entry Q05957) is synthesized in the senescent flower petals and is named the "petal death protein" or "PDP". On the basis of an analysis of the structural contexts of sequence markers common to the C-C bond lyases of the isocitrate lyase/phosphoenolpyruvate mutase superfamily, a substrate screen that employed a (2R)-malate core structure was designed. Accordingly, stereochemically defined C(2)- and C(3)-substituted malates were synthesized and tested as substrates for PDP-catalyzed cleavage of the C(2)-C(3) bond. The screen identified (2R)-ethyl, (3S)-methylmalate, and oxaloacetate [likely to bind as the hydrate, C(2)(OH)(2) gem-diol] as the most active substrates (for each, k(cat)/K(m) = 2 x 10(4) M(-)(1) s(-)(1)). In contrast to the stringent substrate specificities previously observed for the Escherichia coli isocitrate and 2-methylisocitrate lyases, the PDP tolerated hydrogen, methyl, and to a much lesser extent acetate substituents at the C(3) position (S configuration only) and hydoxyl, methyl, ethyl, propyl, and to a much lesser extent isobutyl substituents at C(2) (R configuration only). It is hypothesized that PDP functions in oxalate production in Ca(2+) sequestering and/or in carbon scavenging from alpha-hydroxycarboxylate catabolites during the biochemical transition accompanying petal senescence.

Arabidopsis CDS blastp result: AK059635 [KOME

Lifescience Database Archive (English)

Full Text Available AK059635 001-031-A08 At4g14330.1 phragmoplast-associated kinesin-related protein 2 ...(PAKRP2) identical to cDNA phragmoplast-associated kinesin-related protein 2 (PAKRP2) GI:16973450 6e-18 ...

Identification of the GTPase superfamily in Mycoplasma synoviae and Mycoplasma hyopneumoniae

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Clayton Luiz Borges

2007-01-01

Full Text Available Mycoplasmas are the smallest known prokaryotes with self-replication ability. They are obligate parasites, taking up many molecules of their hosts and acting as pathogens in men, animals, birds and plants. Mycoplasma hyopneumoniae is the infective agent of swine mycoplasmosis and Mycoplasma synoviae is responsible for subclinical upper respiratory infections that may result in airsacculitis and synovitis in chickens and turkeys. These highly infectious organisms present a worldwide distribution and are responsible for major economic problems. Proteins of the GTPase superfamily occur in all domains of life, regulating functions such as protein synthesis, cell cycle and differentiation. Despite their functional diversity, all GTPases are believed to have evolved from a single common ancestor. In this work we have identified mycoplasma GTPases by searching the complete genome databases of Mycoplasma synoviae and Mycoplasma hyopneumoniae, J (non-pathogenic and 7448 (pathogenic strains. Fifteen ORFs encoding predicted GTPases were found in M. synoviae and in the two strains of M. hyopneumoniae. Searches for conserved G domains in GTPases were performed and the sequences were classified into families. The GTPase phylogenetic analysis showed that the subfamilies were well resolved into clades. The presence of GTPases in the three strains suggests the importance of GTPases in 'minimalist' genomes.

External pH modulates EAG superfamily K+ channels through EAG-specific acidic residues in the voltage sensor

Science.gov (United States)

Kazmierczak, Marcin; Zhang, Xiaofei; Chen, Bihan; Mulkey, Daniel K.; Shi, Yingtang; Wagner, Paul G.; Pivaroff-Ward, Kendra; Sassic, Jessica K.; Bayliss, Douglas A.

2013-01-01

The Ether-a-go-go (EAG) superfamily of voltage-gated K+ channels consists of three functionally distinct gene families (Eag, Elk, and Erg) encoding a diverse set of low-threshold K+ currents that regulate excitability in neurons and muscle. Previous studies indicate that external acidification inhibits activation of three EAG superfamily K+ channels, Kv10.1 (Eag1), Kv11.1 (Erg1), and Kv12.1 (Elk1). We show here that Kv10.2, Kv12.2, and Kv12.3 are similarly inhibited by external protons, suggesting that high sensitivity to physiological pH changes is a general property of EAG superfamily channels. External acidification depolarizes the conductance–voltage (GV) curves of these channels, reducing low threshold activation. We explored the mechanism of this high pH sensitivity in Kv12.1, Kv10.2, and Kv11.1. We first examined the role of acidic voltage sensor residues that mediate divalent cation block of voltage activation in EAG superfamily channels because protons reduce the sensitivity of Kv12.1 to Zn2+. Low pH similarly reduces Mg2+ sensitivity of Kv10.1, and we found that the pH sensitivity of Kv11.1 was greatly attenuated at 1 mM Ca2+. Individual neutralizations of a pair of EAG-specific acidic residues that have previously been implicated in divalent block of diverse EAG superfamily channels greatly reduced the pH response in Kv12.1, Kv10.2, and Kv11.1. Our results therefore suggest a common mechanism for pH-sensitive voltage activation in EAG superfamily channels. The EAG-specific acidic residues may form the proton-binding site or alternatively are required to hold the voltage sensor in a pH-sensitive conformation. The high pH sensitivity of EAG superfamily channels suggests that they could contribute to pH-sensitive K+ currents observed in vivo. PMID:23712551

External pH modulates EAG superfamily K+ channels through EAG-specific acidic residues in the voltage sensor.

Science.gov (United States)

Kazmierczak, Marcin; Zhang, Xiaofei; Chen, Bihan; Mulkey, Daniel K; Shi, Yingtang; Wagner, Paul G; Pivaroff-Ward, Kendra; Sassic, Jessica K; Bayliss, Douglas A; Jegla, Timothy

2013-06-01

The Ether-a-go-go (EAG) superfamily of voltage-gated K(+) channels consists of three functionally distinct gene families (Eag, Elk, and Erg) encoding a diverse set of low-threshold K(+) currents that regulate excitability in neurons and muscle. Previous studies indicate that external acidification inhibits activation of three EAG superfamily K(+) channels, Kv10.1 (Eag1), Kv11.1 (Erg1), and Kv12.1 (Elk1). We show here that Kv10.2, Kv12.2, and Kv12.3 are similarly inhibited by external protons, suggesting that high sensitivity to physiological pH changes is a general property of EAG superfamily channels. External acidification depolarizes the conductance-voltage (GV) curves of these channels, reducing low threshold activation. We explored the mechanism of this high pH sensitivity in Kv12.1, Kv10.2, and Kv11.1. We first examined the role of acidic voltage sensor residues that mediate divalent cation block of voltage activation in EAG superfamily channels because protons reduce the sensitivity of Kv12.1 to Zn(2+). Low pH similarly reduces Mg(2+) sensitivity of Kv10.1, and we found that the pH sensitivity of Kv11.1 was greatly attenuated at 1 mM Ca(2+). Individual neutralizations of a pair of EAG-specific acidic residues that have previously been implicated in divalent block of diverse EAG superfamily channels greatly reduced the pH response in Kv12.1, Kv10.2, and Kv11.1. Our results therefore suggest a common mechanism for pH-sensitive voltage activation in EAG superfamily channels. The EAG-specific acidic residues may form the proton-binding site or alternatively are required to hold the voltage sensor in a pH-sensitive conformation. The high pH sensitivity of EAG superfamily channels suggests that they could contribute to pH-sensitive K(+) currents observed in vivo.

CREB binding protein is a required coactivator for Smad-dependent, transforming growth factor β transcriptional responses in endothelial cells

OpenAIRE

Topper, James N.; DiChiara, Maria R.; Brown, Jonathan D.; Williams, Amy J.; Falb, Dean; Collins, Tucker; Gimbrone, Michael A.

1998-01-01

The transforming growth factor-β (TGF-β) superfamily of growth factors and cytokines has been implicated in a variety of physiological and developmental processes within the cardiovascular system. Smad proteins are a recently described family of intracellular signaling proteins that transduce signals in response to TGF-β superfamily ligands. We demonstrate by both a mammalian two-hybrid and a biochemical approach that human Smad2 and Smad4, two essential Smad proteins involved in mediating TG...

Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

Science.gov (United States)

Sugrue, Elena; Fraser, Nicholas J.; Hopkins, Davis H.; Carr, Paul D.; Khurana, Jeevan L.; Oakeshott, John G.; Scott, Colin

2015-01-01

The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible. PMID:25636851

Small GTP-binding proteins in human endothelial cells

NARCIS (Netherlands)

de Leeuw, H. P.; Koster, P. M.; Calafat, J.; Janssen, H.; van Zonneveld, A. J.; van Mourik, J. A.; Voorberg, J.

1998-01-01

Small GTP-binding proteins of the Ras superfamily control an extensive number of intracellular events by alternating between GDP- and GTP-bound conformation. The presence of members of this protein family was examined in human umbilical vein endothelial cells employing RT-PCR. Sequence analysis of

Arabidopsis CDS blastp result: AK287457 [KOME

Lifescience Database Archive (English)

Full Text Available AK287457 J043019L17 At4g14330.1 68417.m02207 phragmoplast-associated kinesin-relate...d protein 2 (PAKRP2) identical to cDNA phragmoplast-associated kinesin-related protein 2 (PAKRP2) GI:16973450 3e-24 ...

Arabidopsis CDS blastp result: AK242767 [KOME

Lifescience Database Archive (English)

Full Text Available AK242767 J090053D16 At4g14330.1 68417.m02207 phragmoplast-associated kinesin-relate...d protein 2 (PAKRP2) identical to cDNA phragmoplast-associated kinesin-related protein 2 (PAKRP2) GI:16973450 1e-20 ...

Arabidopsis CDS blastp result: AK242756 [KOME

Lifescience Database Archive (English)

Full Text Available AK242756 J090051D19 At4g14330.1 68417.m02207 phragmoplast-associated kinesin-relate...d protein 2 (PAKRP2) identical to cDNA phragmoplast-associated kinesin-related protein 2 (PAKRP2) GI:16973450 4e-22 ...

Structural and sequence analysis of imelysin-like proteins implicated in bacterial iron uptake.

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Qingping Xu

Full Text Available Imelysin-like proteins define a superfamily of bacterial proteins that are likely involved in iron uptake. Members of this superfamily were previously thought to be peptidases and were included in the MEROPS family M75. We determined the first crystal structures of two remotely related, imelysin-like proteins. The Psychrobacter arcticus structure was determined at 2.15 Å resolution and contains the canonical imelysin fold, while higher resolution structures from the gut bacteria Bacteroides ovatus, in two crystal forms (at 1.25 Å and 1.44 Å resolution, have a circularly permuted topology. Both structures are highly similar to each other despite low sequence similarity and circular permutation. The all-helical structure can be divided into two similar four-helix bundle domains. The overall structure and the GxHxxE motif region differ from known HxxE metallopeptidases, suggesting that imelysin-like proteins are not peptidases. A putative functional site is located at the domain interface. We have now organized the known homologous proteins into a superfamily, which can be separated into four families. These families share a similar functional site, but each has family-specific structural and sequence features. These results indicate that imelysin-like proteins have evolved from a common ancestor, and likely have a conserved function.

Characterization of the complete mitochondrial genome of Marshallagia marshalli and phylogenetic implications for the superfamily Trichostrongyloidea.

Science.gov (United States)

Sun, Miao-Miao; Han, Liang; Zhang, Fu-Kai; Zhou, Dong-Hui; Wang, Shu-Qing; Ma, Jun; Zhu, Xing-Quan; Liu, Guo-Hua

2018-01-01

Marshallagia marshalli (Nematoda: Trichostrongylidae) infection can lead to serious parasitic gastroenteritis in sheep, goat, and wild ruminant, causing significant socioeconomic losses worldwide. Up to now, the study concerning the molecular biology of M. marshalli is limited. Herein, we sequenced the complete mitochondrial (mt) genome of M. marshalli and examined its phylogenetic relationship with selected members of the superfamily Trichostrongyloidea using Bayesian inference (BI) based on concatenated mt amino acid sequence datasets. The complete mt genome sequence of M. marshalli is 13,891 bp, including 12 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. All protein-coding genes are transcribed in the same direction. Phylogenetic analyses based on concatenated amino acid sequences of the 12 protein-coding genes supported the monophylies of the families Haemonchidae, Molineidae, and Dictyocaulidae with strong statistical support, but rejected the monophyly of the family Trichostrongylidae. The determination of the complete mt genome sequence of M. marshalli provides novel genetic markers for studying the systematics, population genetics, and molecular epidemiology of M. marshalli and its congeners.

A Trypanosoma brucei kinesin heavy chain promotes parasite growth by triggering host arginase activity.

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Géraldine De Muylder

2013-10-01

Full Text Available In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells.By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time.A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.

Evolution of Enzymatic Activities in the Enolase Superfamily: L-Fuconate Dehydratase from Xanthomonas campestris

Energy Technology Data Exchange (ETDEWEB)

Yew,W.; Fedorov, A.; Fedorov, E.; Rakus, J.; Pierce, R.; Almo, S.; Gerlt, J.

2006-01-01

Many members of the mechanistically diverse enolase superfamily have unknown functions. In this report the authors use both genome (operon) context and screening of a library of acid sugars to assign the L-fuconate dehydratase (FucD) function to a member of the mandelate racemase (MR) subgroup of the superfamily encoded by the Xanthomonas campestris pv. campestris str. ATCC 33913 genome (GI: 21233491). Orthologues of FucD are found in both bacteria and eukaryotes, the latter including the rTS beta protein in Homo sapiens that has been implicated in regulating thymidylate synthase activity. As suggested by sequence alignments and confirmed by high-resolution structures in the presence of active site ligands, FucD and MR share the same active site motif of functional groups: three carboxylate ligands for the essential Mg2+ located at the ends of th third, fourth, and fifth-strands in the (/)7-barrel domain (Asp 248, Glu 274, and Glu 301, respectively), a Lys-x-Lys motif at the end of the second-strand (Lys 218 and Lys 220), a His-Asp dyad at the end of the seventh and sixth-strands (His 351 and Asp 324, respectively), and a Glue at the end of the eighth-strand (Glu 382). The mechanism of the FucD reaction involves initial abstraction of the 2-proton by Lys 220, acid catalysis of the vinylogous-elimination of the 3-OH group by His 351, and stereospecific ketonization of the resulting 2-keto-3-deoxy-L-fuconate product. Screening of the library of acid sugars revealed substrate and functional promiscuity: In addition to L-fuconate, FucD also catalyzes the dehydration of L-galactonate, D-arabinonate, D-altronate, L-talonate, and D-ribonate. The dehydrations of L-fuconate, L-galactonate, and D-arabinonate are initiated by abstraction of the 2-protons by Lys 220. The dehydrations of L-talonate and D-ribonate are initiated by abstraction of the 2-protons by His 351; however, protonation of the enediolate intermediates by the conjugate acid of Lys 220 yields L

The 1.8-Å resolution crystal structure of YDR533Cp from Saccharomyces cerevisiae: A member of the DJ-1/ThiJ/PfpI superfamily

Science.gov (United States)

Wilson, Mark A.; Amour, Courtney V. St.; Collins, Jennifer L.; Ringe, Dagmar; Petsko, Gregory A.

2004-01-01

The yeast gene YDR533C encodes a protein belonging to the DJ-1/ThiJ/PfpI superfamily. This family includes the human protein DJ-1, which is mutated in autosomal recessive early-onset Parkinson's disease. The function of DJ-1 and its yeast homologue YDR533Cp is unknown. We report here the crystal structure of YDR533Cp at 1.8-Å resolution. The structure indicates that the closest relative to YDR533Cp is the Escherichia coli heat shock protein Hsp31 (YedU), which has both chaperone and protease activity. As expected, the overall fold of the core domain of YDR533Cp is also similar to that of DJ-1 and the bacterial protease PfpI. YDR533Cp contains a possible catalytic triad analogous to that of Hsp31 and an additional domain that is present in Hsp31 but is not seen in DJ-1 and other members of the family. The cysteine in this triad (Cys-138) is oxidized in this crystal structure, similar to modifications seen in the corresponding cysteine in the crystal structure of DJ-1. YDR533Cp appears to be a dimer both in solution and the crystal, but this dimer is formed by a different interface than that found in Hsp31 or other members of the superfamily. PMID:14745011

BORC/kinesin-1 ensemble drives polarized transport of lysosomes into the axon.

Science.gov (United States)

Farías, Ginny G; Guardia, Carlos M; De Pace, Raffaella; Britt, Dylan J; Bonifacino, Juan S

2017-04-04

The ability of lysosomes to move within the cytoplasm is important for many cellular functions. This ability is particularly critical in neurons, which comprise vast, highly differentiated domains such as the axon and dendrites. The mechanisms that control lysosome movement in these domains, however, remain poorly understood. Here we show that an ensemble of BORC, Arl8, SKIP, and kinesin-1, previously shown to mediate centrifugal transport of lysosomes in nonneuronal cells, specifically drives lysosome transport into the axon, and not the dendrites, in cultured rat hippocampal neurons. This transport is essential for maintenance of axonal growth-cone dynamics and autophagosome turnover. Our findings illustrate how a general mechanism for lysosome dispersal in nonneuronal cells is adapted to drive polarized transport in neurons, and emphasize the importance of this mechanism for critical axonal processes.

Genome-wide identification and analysis of the B3 superfamily of transcription factors in Brassicaceae and major crop plants.

Science.gov (United States)

Peng, Fred Y; Weselake, Randall J

2013-05-01

The plant-specific B3 superfamily of transcription factors has diverse functions in plant growth and development. Using a genome-wide domain analysis, we identified 92, 187, 58, 90, 81, 55, and 77 B3 transcription factor genes in the sequenced genome of Arabidopsis, Brassica rapa, castor bean (Ricinus communis), cocoa (Theobroma cacao), soybean (Glycine max), maize (Zea mays), and rice (Oryza sativa), respectively. The B3 superfamily has substantially expanded during the evolution in eudicots particularly in Brassicaceae, as compared to monocots in the analysis. We observed domain duplication in some of these B3 proteins, forming more complex domain architectures than currently understood. We found that the length of B3 domains exhibits a large variation, which may affect their exact number of α-helices and β-sheets in the core structure of B3 domains, and possibly have functional implications. Analysis of the public microarray data indicated that most of the B3 gene pairs encoding Arabidopsis-rice orthologs are preferentially expressed in different tissues, suggesting their different roles in these two species. Using ESTs in crops, we identified many B3 genes preferentially expressed in reproductive tissues. In a sequence-based quantitative trait loci analysis in rice and maize, we have found many B3 genes associated with traits such as grain yield, seed weight and number, and protein content. Our results provide a framework for future studies into the function of B3 genes in different phases of plant development, especially the ones related to traits in major crops.

Microbial biodegradation of biuret: defining biuret hydrolases within the isochorismatase superfamily.

Science.gov (United States)

Robinson, Serina L; Badalamenti, Jonathan P; Dodge, Anthony G; Tassoulas, Lambros J; Wackett, Lawrence P

2018-03-12

Biuret is a minor component of urea fertilizer and an intermediate in s-triazine herbicide biodegradation. The microbial metabolism of biuret has never been comprehensively studied. Here, we enriched and isolated bacteria from a potato field that grew on biuret as a sole nitrogen source. We sequenced the genome of the fastest-growing isolate, Herbaspirillum sp. BH-1 and identified genes encoding putative biuret hydrolases (BHs). We purified and characterized a functional BH enzyme from Herbaspirillum sp. BH-1 and two other bacteria from divergent phyla. The BH enzymes reacted exclusively with biuret in the range of 2-11 µmol min -1 mg -1 protein. We then constructed a global protein superfamily network to map structure-function relationships in the BH subfamily and used this to mine > 7000 genomes. High-confidence BH sequences were detected in Actinobacteria, Alpha- and Beta-proteobacteria, and some fungi, archaea and green algae, but not animals or land plants. Unexpectedly, no cyanuric acid hydrolase homologs were detected in > 90% of genomes with BH homologs, suggesting BHs may have arisen independently of s-triazine ring metabolism. This work links genotype to phenotype by enabling accurate genome-mining to predict microbial utilization of biuret. Importantly, it advances understanding of the microbial capacity for biuret biodegradation in agricultural systems. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Molecular Characterization and Analysis of a Novel Protein Disulfide Isomerase-Like Protein of Eimeria tenella

OpenAIRE

Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

2014-01-01

Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDI...

Targeting of the tumor necrosis factor receptor superfamily for cancer immunotherapy

NARCIS (Netherlands)

Bremer, Edwin

2013-01-01

The tumor necrosis factor (TNF) ligand and cognate TNF receptor superfamilies constitute an important regulatory axis that is pivotal for immune homeostasis and correct execution of immune responses. TNF ligands and receptors are involved in diverse biological processes ranging from the selective

BORC/kinesin-1 ensemble drives polarized transport of lysosomes into the axon

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Farías, Ginny G.; Guardia, Carlos M.; De Pace, Raffaella; Britt, Dylan J.; Bonifacino, Juan S.

2017-01-01

The ability of lysosomes to move within the cytoplasm is important for many cellular functions. This ability is particularly critical in neurons, which comprise vast, highly differentiated domains such as the axon and dendrites. The mechanisms that control lysosome movement in these domains, however, remain poorly understood. Here we show that an ensemble of BORC, Arl8, SKIP, and kinesin-1, previously shown to mediate centrifugal transport of lysosomes in nonneuronal cells, specifically drives lysosome transport into the axon, and not the dendrites, in cultured rat hippocampal neurons. This transport is essential for maintenance of axonal growth-cone dynamics and autophagosome turnover. Our findings illustrate how a general mechanism for lysosome dispersal in nonneuronal cells is adapted to drive polarized transport in neurons, and emphasize the importance of this mechanism for critical axonal processes. PMID:28320970

Another cat and mouse game: Deciphering the evolution of the SCGB superfamily and exploring the molecular similarity of major cat allergen Fel d 1 and mouse ABP using computational approaches.

Science.gov (United States)

Durairaj, Rajesh; Pageat, Patrick; Bienboire-Frosini, Cécile

2018-01-01

The mammalian secretoglobin (SCGB) superfamily contains functionally diverse members, among which the major cat allergen Fel d 1 and mouse salivary androgen-binding protein (ABP) display similar subunits. We searched for molecular similarities between Fel d 1 and ABP to examine the possibility that they play similar roles. We aimed to i) cluster the evolutionary relationships of the SCGB superfamily; ii) identify divergence patterns, structural overlap, and protein-protein docking between Fel d 1 and ABP dimers; and iii) explore the residual interaction between ABP dimers and steroid binding in chemical communication using computational approaches. We also report that the evolutionary tree of the SCGB superfamily comprises seven unique palm-like clusters, showing the evolutionary pattern and divergence time tree of Fel d 1 with 28 ABP paralogs. Three ABP subunits (A27, BG27, and BG26) share phylogenetic relationships with Fel d 1 chains. The Fel d 1 and ABP subunits show similarities in terms of sequence conservation, identical motifs and binding site clefts. Topologically equivalent positions were visualized through superimposition of ABP A27:BG27 (AB) and ABP A27:BG26 (AG) dimers on a heterodimeric Fel d 1 model. In docking, Fel d 1-ABP dimers exhibit the maximum surface binding ability of AG compared with that of AB dimers and the several polar interactions between ABP dimers with steroids. Hence, cat Fel d 1 is an ABP-like molecule in which monomeric chains 1 and 2 are the equivalent of the ABPA and ABPBG monomers, respectively. These findings suggest that the biological and molecular function of Fel d 1 is similar to that of ABP in chemical communication, possibly via pheromone and/or steroid binding.

Another cat and mouse game: Deciphering the evolution of the SCGB superfamily and exploring the molecular similarity of major cat allergen Fel d 1 and mouse ABP using computational approaches

Science.gov (United States)

Pageat, Patrick; Bienboire-Frosini, Cécile

2018-01-01

The mammalian secretoglobin (SCGB) superfamily contains functionally diverse members, among which the major cat allergen Fel d 1 and mouse salivary androgen-binding protein (ABP) display similar subunits. We searched for molecular similarities between Fel d 1 and ABP to examine the possibility that they play similar roles. We aimed to i) cluster the evolutionary relationships of the SCGB superfamily; ii) identify divergence patterns, structural overlap, and protein-protein docking between Fel d 1 and ABP dimers; and iii) explore the residual interaction between ABP dimers and steroid binding in chemical communication using computational approaches. We also report that the evolutionary tree of the SCGB superfamily comprises seven unique palm-like clusters, showing the evolutionary pattern and divergence time tree of Fel d 1 with 28 ABP paralogs. Three ABP subunits (A27, BG27, and BG26) share phylogenetic relationships with Fel d 1 chains. The Fel d 1 and ABP subunits show similarities in terms of sequence conservation, identical motifs and binding site clefts. Topologically equivalent positions were visualized through superimposition of ABP A27:BG27 (AB) and ABP A27:BG26 (AG) dimers on a heterodimeric Fel d 1 model. In docking, Fel d 1-ABP dimers exhibit the maximum surface binding ability of AG compared with that of AB dimers and the several polar interactions between ABP dimers with steroids. Hence, cat Fel d 1 is an ABP-like molecule in which monomeric chains 1 and 2 are the equivalent of the ABPA and ABPBG monomers, respectively. These findings suggest that the biological and molecular function of Fel d 1 is similar to that of ABP in chemical communication, possibly via pheromone and/or steroid binding. PMID:29771985

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

Science.gov (United States)

Mori, Tetsuya; Saveliev, Sergei V; Xu, Yao; Stafford, Walter F; Cox, Michael M; Inman, Ross B; Johnson, Carl H

2002-12-24

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecADnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

Improving protein fold recognition by extracting fold-specific features from predicted residue-residue contacts.

Science.gov (United States)

Zhu, Jianwei; Zhang, Haicang; Li, Shuai Cheng; Wang, Chao; Kong, Lupeng; Sun, Shiwei; Zheng, Wei-Mou; Bu, Dongbo

2017-12-01

Accurate recognition of protein fold types is a key step for template-based prediction of protein structures. The existing approaches to fold recognition mainly exploit the features derived from alignments of query protein against templates. These approaches have been shown to be successful for fold recognition at family level, but usually failed at superfamily/fold levels. To overcome this limitation, one of the key points is to explore more structurally informative features of proteins. Although residue-residue contacts carry abundant structural information, how to thoroughly exploit these information for fold recognition still remains a challenge. In this study, we present an approach (called DeepFR) to improve fold recognition at superfamily/fold levels. The basic idea of our approach is to extract fold-specific features from predicted residue-residue contacts of proteins using deep convolutional neural network (DCNN) technique. Based on these fold-specific features, we calculated similarity between query protein and templates, and then assigned query protein with fold type of the most similar template. DCNN has showed excellent performance in image feature extraction and image recognition; the rational underlying the application of DCNN for fold recognition is that contact likelihood maps are essentially analogy to images, as they both display compositional hierarchy. Experimental results on the LINDAHL dataset suggest that even using the extracted fold-specific features alone, our approach achieved success rate comparable to the state-of-the-art approaches. When further combining these features with traditional alignment-related features, the success rate of our approach increased to 92.3%, 82.5% and 78.8% at family, superfamily and fold levels, respectively, which is about 18% higher than the state-of-the-art approach at fold level, 6% higher at superfamily level and 1% higher at family level. An independent assessment on SCOP_TEST dataset showed consistent

The UDP glucuronosyltransferase gene superfamily: suggested nomenclature based on evolutionary divergence

NARCIS (Netherlands)

Burchell, B.; Nebert, D. W.; Nelson, D. R.; Bock, K. W.; Iyanagi, T.; Jansen, P. L.; Lancet, D.; Mulder, G. J.; Chowdhury, J. R.; Siest, G.

1991-01-01

A nomenclature system for the UDP glucuronosyltransferase superfamily is proposed, based on divergent evolution of the genes. A total of 26 distinct cDNAs in five mammalian species have been sequenced to date. Comparison of the deduced amino acid sequences leads to the definition of two families and

Relative Stabilities of Conserved and Non-Conserved Structures in the OB-Fold Superfamily

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Andrei T. Alexandrescu

2009-05-01

Full Text Available The OB-fold is a diverse structure superfamily based on a β-barrel motif that is often supplemented with additional non-conserved secondary structures. Previous deletion mutagenesis and NMR hydrogen exchange studies of three OB-fold proteins showed that the structural stabilities of sites within the conserved β-barrels were larger than sites in non-conserved segments. In this work we examined a database of 80 representative domain structures currently classified as OB-folds, to establish the basis of this effect. Residue-specific values were obtained for the number of Cα-Cα distance contacts, sequence hydrophobicities, crystallographic B-factors, and theoretical B-factors calculated from a Gaussian Network Model. All four parameters point to a larger average flexibility for the non-conserved structures compared to the conserved β-barrels. The theoretical B-factors and contact densities show the highest sensitivity.Our results suggest a model of protein structure evolution in which novel structural features develop at the periphery of conserved motifs. Core residues are more resistant to structural changes during evolution since their substitution would disrupt a larger number of interactions. Similar factors are likely to account for the differences in stability to unfolding between conserved and non-conserved structures.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons.

Science.gov (United States)

Niwa, Shinsuke

2017-10-18

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

An Atlas of Peroxiredoxins Created Using an Active Site Profile-Based Approach to Functionally Relevant Clustering of Proteins.

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Angela F Harper

2017-02-01

Full Text Available Peroxiredoxins (Prxs or Prdxs are a large protein superfamily of antioxidant enzymes that rapidly detoxify damaging peroxides and/or affect signal transduction and, thus, have roles in proliferation, differentiation, and apoptosis. Prx superfamily members are widespread across phylogeny and multiple methods have been developed to classify them. Here we present an updated atlas of the Prx superfamily identified using a novel method called MISST (Multi-level Iterative Sequence Searching Technique. MISST is an iterative search process developed to be both agglomerative, to add sequences containing similar functional site features, and divisive, to split groups when functional site features suggest distinct functionally-relevant clusters. Superfamily members need not be identified initially-MISST begins with a minimal representative set of known structures and searches GenBank iteratively. Further, the method's novelty lies in the manner in which isofunctional groups are selected; rather than use a single or shifting threshold to identify clusters, the groups are deemed isofunctional when they pass a self-identification criterion, such that the group identifies itself and nothing else in a search of GenBank. The method was preliminarily validated on the Prxs, as the Prxs presented challenges of both agglomeration and division. For example, previous sequence analysis clustered the Prx functional families Prx1 and Prx6 into one group. Subsequent expert analysis clearly identified Prx6 as a distinct functionally relevant group. The MISST process distinguishes these two closely related, though functionally distinct, families. Through MISST search iterations, over 38,000 Prx sequences were identified, which the method divided into six isofunctional clusters, consistent with previous expert analysis. The results represent the most complete computational functional analysis of proteins comprising the Prx superfamily. The feasibility of this novel method is

The short mRNA isoform of the immunoglobulin superfamily, member 1 gene encodes an intracellular glycoprotein.

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Ying Wang

Full Text Available Mutations in the immunoglobulin superfamily, member 1 gene (IGSF1/Igsf1 cause an X-linked form of central hypothyroidism. The canonical form of IGSF1 is a transmembrane glycoprotein with 12 immunoglobulin (Ig loops. The protein is co-translationally cleaved into two sub-domains. The carboxyl-terminal domain (CTD, which contains the last 7 Ig loops, is trafficked to the plasma membrane. Most pathogenic mutations in IGSF1 map to the portion of the gene encoding the CTD. IGSF1/Igsf1 encodes a variety of transcripts. A little studied, but abundant splice variant encodes a truncated form of the protein, predicted to contain the first 2 Ig loops of the full-length IGSF1. The protein (hereafter referred to as IGSF1 isoform 2 or IGSF1-2 is likely retained in most individuals with IGSF1 mutations. Here, we characterized basic biochemical properties of the protein as a foray into understanding its potential function. IGSF1-2, like the IGSF1-CTD, is a glycoprotein. In both mouse and rat, the protein is N-glycosylated at a single asparagine residue in the first Ig loop. Contrary to earlier predictions, neither the murine nor rat IGSF1-2 is secreted from heterologous or homologous cells. In addition, neither protein associates with the plasma membrane. Rather, IGSF1-2 appears to be retained in the endoplasmic reticulum. Whether the protein plays intracellular functions or is trafficked through the secretory pathway under certain physiologic or pathophysiologic conditions has yet to be determined.

Spatial and temporal expression of immunoglobulin superfamily member 1 in the rat

NARCIS (Netherlands)

Joustra, Sjoerd D.; Meijer, Onno C.; Heinen, Charlotte A.; Mol, Isabel M.; Laghmani, El Houari; Sengers, Rozemarijn M. A.; Carreno, Gabriela; van Trotsenburg, A. S. Paul; Biermasz, Nienke R.; Bernard, Daniel J.; Wit, Jan M.; Oostdijk, Wilma; van Pelt, Ans M. M.; Hamer, Geert; Wagenaar, Gerry T. M.

2015-01-01

Loss-of-function mutations in the immunoglobulin superfamily member 1 (IGSF1) gene cause an X-linked syndrome of central hypothyroidism, macroorchidism, variable prolactin and GH deficiency, delayed pubertal testosterone rise, and obesity. To understand the pathophysiology of this syndrome,

Molecular and Functional Characterization of Mouse S5D-SRCRB: A New Group B Member of the Scavenger Receptor Cysteine-Rich Superfamily

DEFF Research Database (Denmark)

Miró-Julià, Cristina; Roselló, Sandra; Martínez, Vanesa G

2011-01-01

The scavenger receptor cysteine-rich superfamily (SRCR-SF) members are transmembrane and/or secreted receptors exhibiting one or several repeats of a cysteine-rich protein module of ∼100 aa, named scavenger receptor cysteine-rich (SRCR). Two types of SRCR domains (A or B) have been reported, which...... differ in the number of coding exons and intradomain cysteines. Although no unifying function has been reported for SRCR-SF members, recognition of pathogen-associated molecular patterns (PAMPs) was recently shown for some of them. In this article, we report the structural and functional characterization...

Identification of Orch3, a locus controlling dominant resistance to autoimmune orchitis, as kinesin family member 1C.

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Roxana del Rio

Full Text Available Experimental autoimmune orchitis (EAO, the principal model of non-infectious testicular inflammatory disease, can be induced in susceptible mouse strains by immunization with autologous testicular homogenate and appropriate adjuvants. As previously established, the genome of DBA/2J mice encodes genes that are capable of conferring dominant resistance to EAO, while the genome of BALB/cByJ mice does not and they are therefore susceptible to EAO. In a genome scan, we previously identified Orch3 as the major quantitative trait locus controlling dominant resistance to EAO and mapped it to chromosome 11. Here, by utilizing a forward genetic approach, we identified kinesin family member 1C (Kif1c as a positional candidate for Orch3 and, using a transgenic approach, demonstrated that Kif1c is Orch3. Mechanistically, we showed that the resistant Kif1c(D2 allele leads to a reduced antigen-specific T cell proliferative response as a consequence of decreased MHC class II expression by antigen presenting cells, and that the L(578 → P(578 and S(1027 → P(1027 polymorphisms distinguishing the BALB/cByJ and DBA/2J alleles, respectively, can play a role in transcriptional regulation. These findings may provide mechanistic insight into how polymorphism in other kinesins such as KIF21B and KIF5A influence susceptibility and resistance to human autoimmune diseases.

Chemical and thermal modulation of molecular motor activities

Science.gov (United States)

Hong, Weili

Molecular motors of kinesin and dynein families are responsible for various intracellular activities, from long distance movement of organelles, vesicles, protein complexes, and mRNAs to powering mitotic processes. They can take nanometer steps using chemical energy from the hydrolysis of ATP (adenosine triphosphate), and their dysfunction is involved in many neurodegenerative diseases that require long distance transport of cargos. Here I report on the study of the properties of molecular motors at a single-molecule level using optical trappings. I first studied the inhibition properties of kinesin motors by marine natural compound adociasulfates. I showed that adociasulfates compete with microtubules for binding to kinesins and thus inhibit kinesins' activity. Although adociasulfates are a strong inhibitor for all kinesin members, they show a much higher inhibition effect for conventional kinesins than for mitotic kinesins. Thus adociasulfates can be used to specifically inhibit conventional kinesins. By comparing the inhibition of kinesins by two structurally similar adociasulfates, one can see that the negatively charged sulfate residue of adociasulfates can be replaced by other negative residues and thus make it possible for adociasulfate-derived compounds to be more cell permeable. Kinesins and dyneins move cargos towards opposite directions along a microtubule. Cargos with both kinesins and dyneins attached often move bidirectionally due to undergoing a tug-of-war between the oppositely moving kinesin and dynein motors. Here I studied the effect of temperature on microtubule-based kinesin and dynein motor transport. While kinesins' and dyneins' velocities are closely matched above 15 °C, below this temperature the dyneins' velocity decreases much faster than the kinesins'. The kinesins' and dyneins' forces do not measurably change with temperature. The results suggest that temperature has significant effects on bidirectional transport and can be used to

Kinetic and Structural Characterization of a Heterohexamer 4-Oxalocrotonate Tautomerase from Chloroflexus aurantiacus J-10-fl: Implications for Functional and Structural Diversity in the Tautomerase Superfamily

International Nuclear Information System (INIS)

Burks, Elizabeth A.; Fleming, Christopher D.; Mesecar, Andrew D.; Whitman, Christian P.; Pegan, Scott D.

2010-01-01

4-Oxalocrotonate tautomerase (4-OT) isozymes play prominent roles in the bacterial utilization of aromatic hydrocarbons as sole carbon sources. These enzymes catalyze the conversion of 2-hydroxy-2,4-hexadienedioate (or 2-hydroxymuconate) to 2-oxo-3-hexenedioate, where Pro-1 functions as a general base and shuttles a proton from the 2-hydroxyl group of the substrate to the C-5 position of the product. 4-OT, a homohexamer from Pseudomonas putida mt-2, is the most extensively studied 4-OT isozyme and the founding member of the tautomerase superfamily. A search of five thermophilic bacterial genomes identified a coded amino acid sequence in each that had been annotated as a tautomerase-like protein but lacked Pro-1. However, a nearby sequence has Pro-1, but the sequence is not annotated as a tautomerase-like protein. To characterize this group of proteins, two genes from Chloroflexus aurantiacus J-10-fl were cloned, and the corresponding proteins were expressed. Kinetic, biochemical, and X-ray structural analyses show that the two expressed proteins form a functional heterohexamer 4-OT (hh4-OT), composed of three αβ dimers. Like the P. putida enzyme, hh4-OT requires the amino-terminal proline and two arginines for the conversion of 2-hydroxymuconate to the product, implicating an analogous mechanism. In contrast to 4-OT, hh4-OT does not exhibit the low-level activity of another tautomerase superfamily member, the heterohexamer trans-3-chloroacrylic acid dehalogenase (CaaD). Characterization of hh4-OT enables functional assignment of the related enzymes, highlights the diverse ways the β-α-β building block can be assembled into an active enzyme, and provides further insight into the molecular basis of the low-level CaaD activity in 4-OT.

Characterization of a novel wheat endosperm protein belonging to the prolamin superfamily

Science.gov (United States)

Starch granule surface-associated proteins were separated by HPLC and identified by direct protein sequencing. Among the proteins identified was one that consisted of two polypeptide chains of 11 kDa and 19 kDa linked by disulfide bonds. Sequencing of tryptic peptides from each of the polypeptide ch...

The FTO (fat mass and obesity associated gene codes for a novel member of the non-heme dioxygenase superfamily

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Andrade-Navarro Miguel A

2007-11-01

Full Text Available Abstract Background Genetic variants in the FTO (fat mass and obesity associated gene have been associated with an increased risk of obesity. However, the function of its protein product has not been experimentally studied and previously reported sequence similarity analyses suggested the absence of homologs in existing protein databases. Here, we present the first detailed computational analysis of the sequence and predicted structure of the protein encoded by FTO. Results We performed a sequence similarity search using the human FTO protein as query and then generated a profile using the multiple sequence alignment of the homologous sequences. Profile-to-sequence and profile-to-profile based comparisons identified remote homologs of the non-heme dioxygenase family. Conclusion Our analysis suggests that human FTO is a member of the non-heme dioxygenase (Fe(II- and 2-oxoglutarate-dependent dioxygenases superfamily. Amino acid conservation patterns support this hypothesis and indicate that both 2-oxoglutarate and iron should be important for FTO function. This computational prediction of the function of FTO should suggest further steps for its experimental characterization and help to formulate hypothesis about the mechanisms by which it relates to obesity in humans.

Genome-wide identification of nuclear receptor (NR) superfamily genes in the copepod Tigriopus japonicus.

Science.gov (United States)

Hwang, Dae-Sik; Lee, Bo-Young; Kim, Hui-Su; Lee, Min Chul; Kyung, Do-Hyun; Om, Ae-Son; Rhee, Jae-Sung; Lee, Jae-Seong

2014-11-18

Nuclear receptors (NRs) are a large superfamily of proteins defined by a DNA-binding domain (DBD) and a ligand-binding domain (LBD). They function as transcriptional regulators to control expression of genes involved in development, homeostasis, and metabolism. The number of NRs differs from species to species, because of gene duplications and/or lineage-specific gene losses during metazoan evolution. Many NRs in arthropods interact with the ecdysteroid hormone and are involved in ecdysone-mediated signaling in arthropods. The nuclear receptor superfamily complement has been reported in several arthropods, including crustaceans, but not in copepods. We identified the entire NR repertoire of the copepod Tigriopus japonicus, which is an important marine model species for ecotoxicology and environmental genomics. Using whole genome and transcriptome sequences, we identified a total of 31 nuclear receptors in the genome of T. japonicus. Nomenclature of the nuclear receptors was determined based on the sequence similarities of the DNA-binding domain (DBD) and ligand-binding domain (LBD). The 7 subfamilies of NRs separate into five major clades (subfamilies NR1, NR2, NR3, NR4, and NR5/6). Although the repertoire of NR members in, T. japonicus was similar to that reported for other arthropods, there was an expansion of the NR1 subfamily in Tigriopus japonicus. The twelve unique nuclear receptors identified in T. japonicus are members of NR1L. This expansion may be a unique lineage-specific feature of crustaceans. Interestingly, E78 and HR83, which are present in other arthropods, were absent from the genomes of T. japonicus and two congeneric copepod species (T. japonicus and Tigriopus californicus), suggesting copepod lineage-specific gene loss. We identified all NR receptors present in the copepod, T. japonicus. Knowledge of the copepod nuclear receptor repertoire will contribute to a better understanding of copepod- and crustacean-specific NR evolution.

Origination, expansion, evolutionary trajectory, and expression bias of AP2/ERF superfamily in Brassica napus

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Xiaoming Song

2016-08-01

Full Text Available The AP2/ERF superfamily, one of the most important transcription factor families, plays crucial roles in response to biotic and abiotic stresses. So far, a comprehensive evolutionary inference of its origination and expansion has not been available. Here, we identified 515 AP2/ERF genes in B. napus, a neo-tetraploid forming ~7500 years ago, and found that 82.14% of them were duplicated in the tetraploidization. A prominent subgenome bias was revealed in gene expression, tissue-specific, and gene conversion. Moreover, a large-scale analysis across plants and alga suggested that this superfamily could have been originated from AP2 family, expanding to form other families (ERF, and RAV. This process was accompanied by duplicating and/or alternative deleting AP2 domain, intragenic domain sequence conversion, and/or by acquiring other domains, resulting in copy number variations, alternatively contributing to functional innovation. We found that significant positive selection occurred at certain critical nodes during the evolution of land plants, possibly responding to changing environment. In conclusion, the present research revealed origination, functional innovation, and evolutionary trajectory of the AP2/ERF superfamily, contributing to understanding their roles in plant stress tolerance.

Distant relationships amongst protein domains

Indian Academy of Sciences (India)

ncbs

Homologous sequences of individual superfamily members are aligned and amino acid exchanges at individual positions were scored for conservation. Step – 1. Identification of structural motifs. Page 7. Alignment with Homologues. Page 8. il8 like proteins. 0. 50. 100. 150. 200. 250. 1. 6. 11. 16. 21. 26. 31. 36. 41. 46. 51.

At the Perphery of the Amidohydrolase Superfamily: Bh0493 from Bacillus halodurans Catalyzes the Isomerization of D-Galacturonate to D-Tagaturonate

Energy Technology Data Exchange (ETDEWEB)

Nguyen,T.; Brown, S.; Fedorov, A.; Fedorov, E.; Babbitt, P.; Almo, S.; Raushel, F.

2008-01-01

The amidohydrolase superfamily is a functionally diverse set of enzymes that catalyzes predominantly hydrolysis reactions involving sugars, nucleic acids, amino acids, and organophosphate esters. One of the most divergent members of this superfamily, uronate isomerase from Escherichia coli, catalyzes the isomerization of d-glucuronate to d-fructuronate and d-galacturonate to d-tagaturonate and is the only uronate isomerase in this organism. A gene encoding a putative uronate isomerase in Bacillus halodurans (Bh0705) was identified based on sequence similarity to uronate isomerases from other organisms. Kinetic evidence indicates that Bh0705 is relatively specific for the isomerization of d-glucuronate to d-fructuronate, confirming this functional assignment. Despite a low sequence identity to all other characterized uronate isomerases, phylogenetic and network-based analysis suggests that a second gene in this organism, Bh0493, is also a uronate isomerase, although it is an outlier in the group, with <20% sequence identity to any other characterized uronate isomerase from another species. The elucidation of the X-ray structure at a resolution of 2.0 Angstroms confirms that Bh0493 is a member of the amidohydrolase superfamily with conserved residues common to other members of the uronate isomerase family. Functional characterization of this protein shows that unlike Bh0705, Bh0493 can utilize both d-glucuronate and d-galacturonate as substrates. In B. halodurans, Bh0705 is found in an operon for the metabolism of d-glucuronate, whereas Bh0493 is in an operon for the metabolism of d-galacturonate. These results provide the first identification of a uronate isomerase that operates in a pathway distinct from that for d-glucuronate. While most organisms that contain this pathway have only one gene for a uronate isomerase, sequence analysis and operon context show that five other organisms also appear to have two genes and one organism appears to have three genes for

Comparative analysis of cystatin superfamily in platyhelminths.

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Aijiang Guo

Full Text Available The cystatin superfamily is comprised of cysteine proteinase inhibitors and encompasses at least 3 subfamilies: stefins, cystatins and kininogens. In this study, the platyhelminth cystatin superfamily was identified and grouped into stefin and cystatin subfamilies. The conserved domain of stefins (G, QxVxG was observed in all members of platyhelminth stefins. The three characteristics of cystatins, the cystatin-like domain (G, QxVxG, PW, a signal peptide, and one or two conserved disulfide bonds, were observed in platyhelminths, with the exception of cestodes, which lacked the conserved disulfide bond. However, it is noteworthy that cestode cystatins had two tandem repeated domains, although the second tandem repeated domain did not contain a cystatin-like domain, which has not been previously reported. Tertiary structure analysis of Taenia solium cystatin, one of the cestode cystatins, demonstrated that the N-terminus of T. solium cystatin formed a five turn α-helix, a five stranded β-pleated sheet and a hydrophobic edge, similar to the structure of chicken cystatin. Although no conserved disulfide bond was found in T. solium cystatin, the models of T. solium cystatin and chicken cystatin corresponded at the site of the first disulfide bridge of the chicken cystatin. However, the two models were not similar regarding the location of the second disulfide bridge of chicken cystatin. These results showed that T. solium cystatin and chicken cystatin had similarities and differences, suggesting that the biochemistry of T. solium cystatin could be similar to chicken cystatin in its inhibitory function and that it may have further functional roles. The same results were obtained for other cestode cystatins. Phylogenetic analysis showed that cestode cystatins constituted an independent clade and implied that cestode cystatins should be considered to have formed a new clade during evolution.

IGSF9 Family Proteins

DEFF Research Database (Denmark)

Hansen, Maria; Walmod, Peter Schledermann

2013-01-01

The Drosophila protein Turtle and the vertebrate proteins immunoglobulin superfamily (IgSF), member 9 (IGSF9/Dasm1) and IGSF9B are members of an evolutionarily ancient protein family. A bioinformatics analysis of the protein family revealed that invertebrates contain only a single IGSF9 family gene......, the longest isoforms of the proteins have the same general organization as the neural cell adhesion molecule family of cell adhesion molecule proteins, and like this family of proteins, IGSF9 family members are expressed in the nervous system. A review of the literature revealed that Drosophila Turtle...... facilitates homophilic cell adhesion. Moreover, IGSF9 family proteins have been implicated in the outgrowth and branching of neurites, axon guidance, synapse maturation, self-avoidance, and tiling. However, despite the few published studies on IGSF9 family proteins, reports on the functions of both Turtle...

K-nearest uphill clustering in the protein structure space

KAUST Repository

Cui, Xuefeng

2016-08-26

The protein structure classification problem, which is to assign a protein structure to a cluster of similar proteins, is one of the most fundamental problems in the construction and application of the protein structure space. Early manually curated protein structure classifications (e.g., SCOP and CATH) are very successful, but recently suffer the slow updating problem because of the increased throughput of newly solved protein structures. Thus, fully automatic methods to cluster proteins in the protein structure space have been designed and developed. In this study, we observed that the SCOP superfamilies are highly consistent with clustering trees representing hierarchical clustering procedures, but the tree cutting is very challenging and becomes the bottleneck of clustering accuracy. To overcome this challenge, we proposed a novel density-based K-nearest uphill clustering method that effectively eliminates noisy pairwise protein structure similarities and identifies density peaks as cluster centers. Specifically, the density peaks are identified based on K-nearest uphills (i.e., proteins with higher densities) and K-nearest neighbors. To our knowledge, this is the first attempt to apply and develop density-based clustering methods in the protein structure space. Our results show that our density-based clustering method outperforms the state-of-the-art clustering methods previously applied to the problem. Moreover, we observed that computational methods and human experts could produce highly similar clusters at high precision values, while computational methods also suggest to split some large superfamilies into smaller clusters. © 2016 Elsevier B.V.

GH97 is a new family of glycoside hydrolases, which is related to the α-galactosidase superfamily

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Naumoff Daniil G

2005-08-01

Full Text Available Abstract Background As a rule, about 1% of genes in a given genome encode glycoside hydrolases and their homologues. On the basis of sequence similarity they have been grouped into more than ninety GH families during the last 15 years. The GH97 family has been established very recently and initially included only 18 bacterial proteins. However, the evolutionary relationship of the genes encoding proteins of this family remains unclear, as well as their distribution among main groups of the living organisms. Results The extensive search of the current databases allowed us to double the number of GH97 family proteins. Five subfamilies were distinguished on the basis of pairwise sequence comparison and phylogenetic analysis. Iterative sequence analysis revealed the relationship of the GH97 family with the GH27, GH31, and GH36 families of glycosidases, which belong to the α-galactosidase superfamily, as well as a more distant relationship with some other glycosidase families (GH13 and GH20. Conclusion The results of this study show an unexpected sequence similarity of GH97 family proteins with glycoside hydrolases from several other families, that have (β/α8-barrel fold of the catalytic domain and a retaining mechanism of the glycoside bond hydrolysis. These data suggest a common evolutionary origin of glycosidases representing different families and clans.

Discovery of a distinct superfamily of Kunitz-type toxin (KTT from tarantulas.

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Chun-Hua Yuan

Full Text Available BACKGROUND: Kuntiz-type toxins (KTTs have been found in the venom of animals such as snake, cone snail and sea anemone. The main ancestral function of Kunitz-type proteins was the inhibition of a diverse array of serine proteases, while toxic activities (such as ion-channel blocking were developed under a variety of Darwinian selection pressures. How new functions were grafted onto an old protein scaffold and what effect Darwinian selection pressures had on KTT evolution remains a puzzle. PRINCIPAL FINDINGS: Here we report the presence of a new superfamily of ktts in spiders (TARANTULAS: Ornithoctonus huwena and Ornithoctonus hainana, which share low sequence similarity to known KTTs and is clustered in a distinct clade in the phylogenetic tree of KTT evolution. The representative molecule of spider KTTs, HWTX-XI, purified from the venom of O. huwena, is a bi-functional protein which is a very potent trypsin inhibitor (about 30-fold more strong than BPTI as well as a weak Kv1.1 potassium channel blocker. Structural analysis of HWTX-XI in 3-D by NMR together with comparative function analysis of 18 expressed mutants of this toxin revealed two separate sites, corresponding to these two activities, located on the two ends of the cone-shape molecule of HWTX-XI. Comparison of non-synonymous/synonymous mutation ratios (omega for each site in spider and snake KTTs, as well as PBTI like body Kunitz proteins revealed high Darwinian selection pressure on the binding sites for Kv channels and serine proteases in snake, while only on the proteases in spider and none detected in body proteins, suggesting different rates and patterns of evolution among them. The results also revealed a series of key events in the history of spider KTT evolution, including the formation of a novel KTT family (named sub-Kuntiz-type toxins derived from the ancestral native KTTs with the loss of the second disulfide bridge accompanied by several dramatic sequence modifications

The superfamily of C3b/C4b-binding proteins

DEFF Research Database (Denmark)

Kristensen, Torsten; D'Eustachio, P; Ogata, R T

1987-01-01

The determination of primary structures by amino acid and nucleotide sequencing for the C3b-and/or C4b-binding proteins H, C4BP, CR1, B, and C2 has revealed the presence of a common structural element. This element is approximately 60 amino acids long and is repeated in a tandem fashion, commencing...... at the amino-terminal end of each molecule. Two other complement components, C1r and C1s, have two of these repeating units in the carboxy-terminal region of their noncatalytic A chains. Three noncomplement proteins, beta 2-glycoprotein I (beta 2I), the interleukin 2 receptor (IL 2 receptor), and the b chain...... of factor XIII, have 4, 2 and 10 of these repeating units, respectively. These proteins obviously belong to the above family, although there is no evidence that they interact with C3b and/or C4b. Human haptoglobin and rat leukocyte common antigen also contain two and three repeating units, respectively...

The Association between Gene-Environment Interactions and Diseases Involving the Human GST Superfamily with SNP Variants

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Antoinesha L. Hollman

2016-03-01

Full Text Available Exposure to environmental hazards has been associated with diseases in humans. The identification of single nucleotide polymorphisms (SNPs in human populations exposed to different environmental hazards, is vital for detecting the genetic risks of some important human diseases. Several studies in this field have been conducted on glutathione S-transferases (GSTs, a phase II detoxification superfamily, to investigate its role in the occurrence of diseases. Human GSTs consist of cytosolic and microsomal superfamilies that are further divided into subfamilies. Based on scientific search engines and a review of the literature, we have found a large amount of published articles on human GST super- and subfamilies that have greatly assisted in our efforts to examine their role in health and disease. Because of its polymorphic variations in relation to environmental hazards such as air pollutants, cigarette smoke, pesticides, heavy metals, carcinogens, pharmaceutical drugs, and xenobiotics, GST is considered as a significant biomarker. This review examines the studies on gene-environment interactions related to various diseases with respect to single nucleotide polymorphisms (SNPs found in the GST superfamily. Overall, it can be concluded that interactions between GST genes and environmental factors play an important role in human diseases.

Hypothesis: NDL proteins function in stress responses by regulating microtubule organization

OpenAIRE

Khatri, Nisha; Mudgil, Yashwanti

2015-01-01

N-MYC DOWNREGULATED-LIKE proteins (NDL), members of the alpha/beta hydrolase superfamily were recently rediscovered as interactors of G-protein signaling in Arabidopsis thaliana. Although the precise molecular function of NDL proteins is still elusive, in animals these proteins play protective role in hypoxia and expression is induced by hypoxia and nickel, indicating role in stress. Homology of NDL1 with animal counterpart N-MYC DOWNREGULATED GENE (NDRG) suggests similar functions in animals...

Phylogenetic comparison of F-Box (FBX gene superfamily within the plant kingdom reveals divergent evolutionary histories indicative of genomic drift.

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Zhihua Hua

Full Text Available The emergence of multigene families has been hypothesized as a major contributor to the evolution of complex traits and speciation. To help understand how such multigene families arose and diverged during plant evolution, we examined the phylogenetic relationships of F-Box (FBX genes, one of the largest and most polymorphic superfamilies known in the plant kingdom. FBX proteins comprise the target recognition subunit of SCF-type ubiquitin-protein ligases, where they individually recruit specific substrates for ubiquitylation. Through the extensive analysis of 10,811 FBX loci from 18 plant species, ranging from the alga Chlamydomonas reinhardtii to numerous monocots and eudicots, we discovered strikingly diverse evolutionary histories. The number of FBX loci varies widely and appears independent of the growth habit and life cycle of land plants, with a little as 198 predicted for Carica papaya to as many as 1350 predicted for Arabidopsis lyrata. This number differs substantially even among closely related species, with evidence for extensive gains/losses. Despite this extraordinary inter-species variation, one subset of FBX genes was conserved among most species examined. Together with evidence of strong purifying selection and expression, the ligases synthesized from these conserved loci likely direct essential ubiquitylation events. Another subset was much more lineage specific, showed more relaxed purifying selection, and was enriched in loci with little or no evidence of expression, suggesting that they either control more limited, species-specific processes or arose from genomic drift and thus may provide reservoirs for evolutionary innovation. Numerous FBX loci were also predicted to be pseudogenes with their numbers tightly correlated with the total number of FBX genes in each species. Taken together, it appears that the FBX superfamily has independently undergone substantial birth/death in many plant lineages, with its size and rapid

Comparative genomic study of ALDH gene superfamily in Gossypium: A focus on Gossypium hirsutum under salt stress.

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Yating Dong

Full Text Available Aldehyde dehydrogenases (ALDHs are a superfamily of enzymes which play important role in the scavenging of active aldehydes molecules. In present work, a comprehensive whole-genomic study of ALDH gene superfamily was carried out for an allotetraploid cultivated cotton species, G. hirsutum, as well as in parallel relative to their diploid progenitors, G. arboreum and G. raimondii. Totally, 30 and 58 ALDH gene sequences belong to 10 families were identified from diploid and allotetraploid cotton species, respectively. The gene structures among the members from same families were highly conserved. Whole-genome duplication and segmental duplication might be the major driver for the expansion of ALDH gene superfamily in G. hirsutum. In addition, the expression patterns of GhALDH genes were diverse across tissues. Most GhALDH genes were induced or repressed by salt stress in upland cotton. Our observation shed lights on the molecular evolutionary properties of ALDH genes in diploid cottons and their alloallotetraploid derivatives. It may be useful to mine key genes for improvement of cotton response to salt stress.

Kif13b Regulates PNS and CNS Myelination through the Dlg1 Scaffold.

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Roberta Noseda

2016-04-01

Full Text Available Microtubule-based kinesin motors have many cellular functions, including the transport of a variety of cargos. However, unconventional roles have recently emerged, and kinesins have also been reported to act as scaffolding proteins and signaling molecules. In this work, we further extend the notion of unconventional functions for kinesin motor proteins, and we propose that Kif13b kinesin acts as a signaling molecule regulating peripheral nervous system (PNS and central nervous system (CNS myelination. In this process, positive and negative signals must be tightly coordinated in time and space to orchestrate myelin biogenesis. Here, we report that in Schwann cells Kif13b positively regulates myelination by promoting p38γ mitogen-activated protein kinase (MAPK-mediated phosphorylation and ubiquitination of Discs large 1 (Dlg1, a known brake on myelination, which downregulates the phosphatidylinositol 3-kinase (PI3K/v-AKT murine thymoma viral oncogene homolog (AKT pathway. Interestingly, Kif13b also negatively regulates Dlg1 stability in oligodendrocytes, in which Dlg1, in contrast to Schwann cells, enhances AKT activation and promotes myelination. Thus, our data indicate that Kif13b is a negative regulator of CNS myelination. In summary, we propose a novel function for the Kif13b kinesin in glial cells as a key component of the PI3K/AKT signaling pathway, which controls myelination in both PNS and CNS.

TGF-β superfamily signaling in testis formation and early male germline development.

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Young, Julia C; Wakitani, Shoichi; Loveland, Kate L

2015-09-01

The TGF-β ligand superfamily contains at least 40 members, many of which are produced and act within the mammalian testis to facilitate formation of sperm. Their progressive expression at key stages and in specific cell types determines the fertility of adult males, influencing testis development and controlling germline differentiation. BMPs are essential for the interactive instructions between multiple cell types in the early embryo that drive initial specification of gamete precursors. In the nascent foetal testis, several ligands including Nodal, TGF-βs, Activins and BMPs, serve as key masculinizing switches by regulating male germline pluripotency, somatic and germline proliferation, and testicular vascularization and architecture. In postnatal life, local production of these factors determine adult testis size by regulating Sertoli cell multiplication and differentiation, in addition to specifying germline differentiation and multiplication. Because TGF-β superfamily signaling is integral to testis formation, it affects processes that underlie testicular pathologies, including testicular cancer, and its potential to contribute to subfertility is beginning to be understood. Copyright © 2015 Elsevier Ltd. All rights reserved.

A cAMP/PKA/Kinesin-1 Axis Promotes the Axonal Transport of Mitochondria in Aging Drosophila Neurons.

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Vagnoni, Alessio; Bullock, Simon L

2018-04-23

Mitochondria play fundamental roles within cells, including energy provision, calcium homeostasis, and the regulation of apoptosis. The transport of mitochondria by microtubule-based motors is critical for neuronal structure and function. This process allows local requirements for mitochondrial functions to be met and also facilitates recycling of these organelles [1, 2]. An age-related reduction in mitochondrial transport has been observed in neurons of mammalian and non-mammalian organisms [3-6], and has been proposed to contribute to the broader decline in neuronal function that occurs during aging [3, 5-7]. However, the factors that influence mitochondrial transport in aging neurons are poorly understood. Here we provide evidence using the tractable Drosophila wing nerve system that the cyclic AMP/protein kinase A (cAMP/PKA) pathway promotes the axonal transport of mitochondria in adult neurons. The level of the catalytic subunit of PKA decreases during aging, and acute activation of the cAMP/PKA pathway in aged flies strongly stimulates mitochondrial motility. Thus, the age-related impairment of transport is reversible. The expression of many genes is increased by PKA activation in aged flies. However, our results indicate that elevated mitochondrial transport is due in part to upregulation of the heavy chain of the kinesin-1 motor, the level of which declines during aging. Our study identifies evolutionarily conserved factors that can strongly influence mitochondrial motility in aging neurons. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

FERM proteins in animal morphogenesis.

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Tepass, Ulrich

2009-08-01

Proteins containing a FERM domain are ubiquitous components of the cytocortex of animal cells where they are engaged in structural, transport, and signaling functions. Recent years have seen a wealth of genetic studies in model organisms that explore FERM protein function in development and tissue organization. In addition, mutations in several FERM protein-encoding genes have been associated with human diseases. This review will provide a brief overview of the FERM domain structure and the FERM protein superfamily and then discuss recent advances in our understanding of the mechanism of function and developmental requirement of several FERM proteins including Moesin, Myosin-VIIA, Myosin-XV, Coracle/Band4.1 as well as Yurt and its vertebrate homologs Mosaic Eyes and EPB41L5/YMO1/Limulus.

A Major Facilitator Superfamily protein encoded by TcMucK gene is not required for cuticle pigmentation, growth and development in Tribolium castaneum.

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Mun, Seulgi; Noh, Mi Young; Osanai-Futahashi, Mizuko; Muthukrishnan, Subbaratnam; Kramer, Karl J; Arakane, Yasuyuki

2014-06-01

Insect cuticle pigmentation and sclerotization (tanning) are vital physiological processes for insect growth, development and survival. We have previously identified several colorless precursor molecules as well as enzymes involved in their biosynthesis and processing to yield the mature intensely colored body cuticle pigments. A recent study indicated that the Bombyx mori (silkmoth) gene, BmMucK, which encodes a protein orthologous to a Culex pipiens quiquefasciatus (Southern house mosquito) cis,cis, muconate transporter, is a member of the "Major Facilitator Superfamily" (MFS) of transporter proteins and is associated with the appearance of pigmented body segments of naturally occurring body color mutants of B. mori. While RNA interference of the BmMucK gene failed to result in any observable phenotype, RNAi using a dsRNA for an orthologous gene from the red flour beetle, Tribolium castaneum, was reported to result in molting defects and darkening of the cuticle and some body parts, leading to the suggestion that orthologs of MucK genes may differ in their functions among insects. To verify the role and essentiality of the ortholog of this gene in development and body pigmentation function in T. castaneum we obtained cDNAs for the orthologous gene (TcMucK) from RNA isolated from the GA-1 wild-type strain of T. castaneum. The sequence of a 1524 nucleotides-long cDNA for TcMucK which encodes the putatively full-length protein, was assembled from two overlapping RT-PCR fragments and the expression profile of this gene during development was analyzed by real-time PCR. This cDNA encodes a 55.8 kDa protein consisting of 507 amino acid residues and includes 11 putative transmembrane segments. Transcripts of TcMucK were detected throughout all of the developmental stages analyzed. The function of this gene was explored by injection of two different double-stranded RNAs targeting different regions of the TcMucK gene (dsTcMucKs) into young larvae to down

Mouse RC/BTB2, a Member of the RCC1 Superfamily, Localizes to Spermatid Acrosomal Vesicles

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Shen, Xuening; Nagarkatti-Gude, David R.; Hess, Rex A.; Henderson, Scott C.; Strauss, Jerome F.; Zhang, Zhibing

2012-01-01

Mouse RC/BTB2 is an unstudied protein of the RCC1 (Regulator of Chromosome Condensation) superfamily. Because of the significant remodeling of chromatin that occurs during spermiogenesis, we characterized the expression and localization of mouse RC/BTB2 in the testis and male germ cells. The Rc/btb2 gene yields two major transcripts: 2.3 kb Rc/btb2-s, present in most somatic tissues examined; and 2.5 kb Rc/btb2-t, which contains a unique non-translated exon in its 5′-UTR that is only detected in the testis. During the first wave of spermatogenesis, Rc/btb2-t mRNA is expressed from day 8 after birth, reaching highest levels of expression at day 30 after birth. The full-length protein contains three RCC1 domains in the N-terminus, and a BTB domain in the C-terminus. In the testis, the protein is detectable from day 12, but is progressively up-regulated to day 30 and day 42 after birth. In spermatids, some of the protein co-localizes with acrosomal markers sp56 and peanut lectin, indicating that it is an acrosomal protein. A GFP-tagged RCC1 domain is present throughout the cytoplasm of transfected CHO cells. However, both GFP-tagged, full-length RC/BTB2 and a GFP-tagged BTB domain localize to vesicles in close proximity to the nuclear membrane, suggesting that the BTB domain might play a role in mediating full-length RC/BTB2 localization. Since RCC1 domains associate with Ran, a small GTPase that regulates molecular trafficking, it is possible that RC/BTB2 plays a role in transporting proteins during acrosome formation. PMID:22768142

Role of multidrug resistance protein (MRP) in glutathione S-conjugate transport in mammalian cells

NARCIS (Netherlands)

Müller, M.; de Vries, E. G.; Jansen, P. L.

1996-01-01

The human multidrug resistance protein (MRP), a 190-kDa member of the ABC-protein superfamily, is an ATP-dependent glutathione S-conjugate carrier (GS-X pump) and is present in membranes of many, if not all, cells. Overexpression of MRP in tumor cells contributes to resistance to natural product

Role of multidrug resistance protein (MRP) in glutathione S-conjugate transport in mammalian cells

NARCIS (Netherlands)

Muller, M; deVries, EGE; Jansen, PLM

1996-01-01

The human multidrug resistance protein (MRP), a 190-kDa member of the ABC-protein superfamily, is an ATP-dependent glutathione S-conjugate carrier (GS-X pump) and is present in membranes of many, if not all, cells, Overexpression of MRP in tumor cells contributes to resistance to natural product

A Novel Superfamily of Transporters for Allantoin and Other Oxo Derivatives of Nitrogen Heterocyclic Compounds in Arabidopsis

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Desimone, Marcelo; Catoni, Elisabetta; Ludewig, Uwe; Hilpert, Melanie; Schneider, Anja; Kunze, Reinhard; Tegeder, Mechthild; Frommer, Wolf Bernd; Schumacher, Karin

2002-01-01

A wide spectrum of soil heterocyclic nitrogen compounds are potential nutrients for plants. Here, it is shown that Arabidopsis plants are able to use allantoin as sole nitrogen source. By functional complementation of a yeast mutant defective in allantoin uptake, an Arabidopsis transporter, AtUPS1 (Arabidopsis thaliana ureide permease 1), was identified. AtUPS1 belongs to a novel superfamily of plant membrane proteins with five open reading frames in Arabidopsis (identity, 64 to 82%). UPS proteins have 10 putative transmembrane domains with a large cytosolic central domain containing a “Walker A” motif. Transport of 14C-labeled allantoin by AtUPS1 in yeast exhibited saturation kinetics (Km ∼ 52 μM), was dependent on Glc and a proton gradient, and was stimulated by acidic pH. AtUPS1 transports uric acid and xanthine, besides allantoin, but not adenine. Protons are cosubstrates in allantoin transport by AtUPS1, as demonstrated by expression in Xenopus laevis oocytes. In plants, AtUPS1 gene expression was dependent on the nitrogen source. Therefore, AtUPS1 presumably is involved in the uptake of allantoin and other purine degradation products when primary sources are limiting. PMID:11971139

Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella.

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Hongyu Han

Full Text Available Protein disulfide isomerase (PDI and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE according to the expressed sequence tag (EST. The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC. BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells

Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella.

Science.gov (United States)

Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

2014-01-01

Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC). BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells. These results

Evolution of Enzymatic Activities in the Enolase Superfamily: L-Rhamnonate Dehydratase

Energy Technology Data Exchange (ETDEWEB)

Rakus,J.; Fedorov, A.; Fedorov, E.; Glaner, M.; Hubbard, B.; Delli, J.; Babbitt, P.; Almo, S.; Gerlt, J.

2008-01-01

The l-rhamnonate dehydratase (RhamD) function was assigned to a previously uncharacterized family in the mechanistically diverse enolase superfamily that is encoded by the genome of Escherichia coli K-12. We screened a library of acid sugars to discover that the enzyme displays a promiscuous substrate specificity: l-rhamnonate (6-deoxy-l-mannonate) has the 'best' kinetic constants, with l-mannonate, l-lyxonate, and d-gulonate dehydrated less efficiently. Crystal structures of the RhamDs from both E. coli K-12 and Salmonella typhimurium LT2 (95% sequence identity) were obtained in the presence of Mg2+; the structure of the RhamD from S. typhimurium was also obtained in the presence of 3-deoxy-l-rhamnonate (obtained by reduction of the product with NaBH4). Like other members of the enolase superfamily, RhamD contains an N-terminal a + {beta} capping domain and a C-terminal ({beta}/a)7{beta}-barrel (modified TIM-barrel) catalytic domain with the active site located at the interface between the two domains. In contrast to other members, the specificity-determining '20s loop' in the capping domain is extended in length and the '50s loop' is truncated. The ligands for the Mg2+ are Asp 226, Glu 252 and Glu 280 located at the ends of the third, fourth and fifth {beta}-strands, respectively. The active site of RhamD contains a His 329-Asp 302 dyad at the ends of the seventh and sixth {beta}-strands, respectively, with His 329 positioned to function as the general base responsible for abstraction of the C2 proton of l-rhamnonate to form a Mg2+-stabilized enediolate intermediate. However, the active site does not contain other acid/base catalysts that have been implicated in the reactions catalyzed by other members of the MR subgroup of the enolase superfamily. Based on the structure of the liganded complex, His 329 also is expected to function as the general acid that both facilitates departure of the 3-OH group in a syn-dehydration reaction and

Regulation of microtubule-based transport by MAP4

Science.gov (United States)

Semenova, Irina; Ikeda, Kazuho; Resaul, Karim; Kraikivski, Pavel; Aguiar, Mike; Gygi, Steven; Zaliapin, Ilya; Cowan, Ann; Rodionov, Vladimir

2014-01-01

Microtubule (MT)-based transport of organelles driven by the opposing MT motors kinesins and dynein is tightly regulated in cells, but the underlying molecular mechanisms remain largely unknown. Here we tested the regulation of MT transport by the ubiquitous protein MAP4 using Xenopus melanophores as an experimental system. In these cells, pigment granules (melanosomes) move along MTs to the cell center (aggregation) or to the periphery (dispersion) by means of cytoplasmic dynein and kinesin-2, respectively. We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs. Overexpression of XMAP4 inhibited pigment aggregation by shortening dynein-dependent MT runs of melanosomes, whereas removal of XMAP4 from MTs reduced the length of kinesin-2–dependent runs and suppressed pigment dispersion. We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2–based movement. Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2–dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect. PMID:25143402

The kinesin-3 family motor KLP-4 regulates anterograde trafficking of GLR-1 glutamate receptors in the ventral nerve cord of Caenorhabditis elegans.

Science.gov (United States)

Monteiro, Michael I; Ahlawat, Shikha; Kowalski, Jennifer R; Malkin, Emily; Koushika, Sandhya P; Juo, Peter

2012-09-01

The transport of glutamate receptors from the cell body to synapses is essential during neuronal development and may contribute to the regulation of synaptic strength in the mature nervous system. We previously showed that cyclin-dependent kinase-5 (CDK-5) positively regulates the abundance of GLR-1 glutamate receptors at synapses in the ventral nerve cord (VNC) of Caenorhabditis elegans. Here we identify a kinesin-3 family motor klp-4/KIF13 in a cdk-5 suppressor screen for genes that regulate GLR-1 trafficking. klp-4 mutants have decreased abundance of GLR-1 in the VNC. Genetic analysis of klp-4 and the clathrin adaptin unc-11/AP180 suggests that klp-4 functions before endocytosis in the ventral cord. Time-lapse microscopy indicates that klp-4 mutants exhibit decreased anterograde flux of GLR-1. Genetic analysis of cdk-5 and klp-4 suggests that they function in the same pathway to regulate GLR-1 in the VNC. Interestingly, GLR-1 accumulates in cell bodies of cdk-5 but not klp-4 mutants. However, GLR-1 does accumulate in klp-4-mutant cell bodies if receptor degradation in the multivesicular body/lysosome pathway is blocked. This study identifies kinesin KLP-4 as a novel regulator of anterograde glutamate receptor trafficking and reveals a cellular control mechanism by which receptor cargo is targeted for degradation in the absence of its motor.

Membrane re-modelling by BAR domain superfamily proteins via molecular and non-molecular factors.

Science.gov (United States)

Nishimura, Tamako; Morone, Nobuhiro; Suetsugu, Shiro

2018-04-17

Lipid membranes are structural components of cell surfaces and intracellular organelles. Alterations in lipid membrane shape are accompanied by numerous cellular functions, including endocytosis, intracellular transport, and cell migration. Proteins containing Bin-Amphiphysin-Rvs (BAR) domains (BAR proteins) are unique, because their structures correspond to the membrane curvature, that is, the shape of the lipid membrane. BAR proteins present at high concentration determine the shape of the membrane, because BAR domain oligomers function as scaffolds that mould the membrane. BAR proteins co-operate with various molecular and non-molecular factors. The molecular factors include cytoskeletal proteins such as the regulators of actin filaments and the membrane scission protein dynamin. Lipid composition, including saturated or unsaturated fatty acid tails of phospholipids, also affects the ability of BAR proteins to mould the membrane. Non-molecular factors include the external physical forces applied to the membrane, such as tension and friction. In this mini-review, we will discuss how the BAR proteins orchestrate membrane dynamics together with various molecular and non-molecular factors. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Short interspersed elements (SINEs) of the Geomyoidea superfamily rodents.

Science.gov (United States)

Gogolevsky, Konstantin P; Kramerov, Dmitri A

2006-05-24

A new short interspersed element (SINE) was isolated from the genome of desert kangaroo rat (Dipodomys deserti) using single-primer PCR. This SINE consists of two monomers: the left monomer (IDL) resembles rodent ID element and other tRNAAla(CGC)-derived SINEs, whereas the right one (Geo) shows no similarity with known SINE sequences. PCR and hybridization analyses demonstrated that IDL-Geo SINE is restricted to the rodent superfamily Geomyoidea (families Geomyidea and Heteromyidea). Isolation and analysis of IDL-Geo from California pocket mouse (Chaetodipus californicus) and Botta's pocket gopher (Thomomys bottae) revealed some species-specific features of this SINE family. The structure and evolution of known dimeric SINEs are discussed.

Analysis of the active site mechanism of Tyrosyl-DNA phosphodiesterase I: a member of the phospholipase D superfamily

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Gajewski, Stefan; Comeaux, Evan Q.; Jafari, Nauzanene; Bharatham, Nagakumar; Bashford, Donald; White, Stephen W.; van Waardenburg, Robert C.A.M.

2011-01-01

Tyrosyl DNA phosphodiesterase I (Tdp1) is a member of the phospholipase D superfamily and hydrolyzes 3′phospho-DNA adducts via two conserved catalytic histidines, one acting as the lead nucleophile and the second as a general acid/base. Substitution of the second histidine specifically to arginine contributes to the neurodegenerative disease SCAN1. We investigated the catalytic role of this histidine in the yeast protein (His432) using a combination of X-ray crystallography, biochemistry, yeast genetics and theoretical chemistry. The structures of wild type Tdp1 and His432Arg both show a phosphorylated form of the nucleophilic histidine that is not observed in the structure of His432Asn. The phosphohistidine is stabilized in the His432Arg structure by the guanidinium group that also restricts access of a nucleophilic water molecule to the Tdp1-DNA intermediate. Biochemical analyses confirm that His432Arg forms an observable and unique Tdp1-DNA adduct during catalysis. Substitution of His432 by Lys does not affect catalytic activity or yeast phenotype, but substitution with Asn, Gln, Leu, Ala, Ser and Thr all result in severely compromised enzymes and Top1-camptothecin dependent lethality. Surprisingly, His432Asn did not show a stable covalent Tdp1-DNA intermediate which suggests another catalytic defect. Theoretical calculations revealed that the defect resides in the nucleophilic histidine and that the pKa of this histidine is crucially dependent upon the second histidine and the incoming phosphate of the substrate. This represents a unique example of substrate-activated catalysis that applies to the entire phospholipase D superfamily. PMID:22155078

Aldehyde Dehydrogenase Gene Superfamily in Populus: Organization and Expression Divergence between Paralogous Gene Pairs.

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Tian, Feng-Xia; Zang, Jian-Lei; Wang, Tan; Xie, Yu-Li; Zhang, Jin; Hu, Jian-Jun

2015-01-01

Aldehyde dehydrogenases (ALDHs) constitute a superfamily of NAD(P)+-dependent enzymes that catalyze the irreversible oxidation of a wide range of reactive aldehydes to their corresponding nontoxic carboxylic acids. ALDHs have been studied in many organisms from bacteria to mammals; however, no systematic analyses incorporating genome organization, gene structure, expression profiles, and cis-acting elements have been conducted in the model tree species Populus trichocarpa thus far. In this study, a comprehensive analysis of the Populus ALDH gene superfamily was performed. A total of 26 Populus ALDH genes were found to be distributed across 12 chromosomes. Genomic organization analysis indicated that purifying selection may have played a pivotal role in the retention and maintenance of PtALDH gene families. The exon-intron organizations of PtALDHs were highly conserved within the same family, suggesting that the members of the same family also may have conserved functionalities. Microarray data and qRT-PCR analysis indicated that most PtALDHs had distinct tissue-specific expression patterns. The specificity of cis-acting elements in the promoter regions of the PtALDHs and the divergence of expression patterns between nine paralogous PtALDH gene pairs suggested that gene duplications may have freed the duplicate genes from the functional constraints. The expression levels of some ALDHs were up- or down-regulated by various abiotic stresses, implying that the products of these genes may be involved in the adaptation of Populus to abiotic stresses. Overall, the data obtained from our investigation contribute to a better understanding of the complexity of the Populus ALDH gene superfamily and provide insights into the function and evolution of ALDH gene families in vascular plants.

Aldehyde Dehydrogenase Gene Superfamily in Populus: Organization and Expression Divergence between Paralogous Gene Pairs.

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Feng-Xia Tian

Full Text Available Aldehyde dehydrogenases (ALDHs constitute a superfamily of NAD(P+-dependent enzymes that catalyze the irreversible oxidation of a wide range of reactive aldehydes to their corresponding nontoxic carboxylic acids. ALDHs have been studied in many organisms from bacteria to mammals; however, no systematic analyses incorporating genome organization, gene structure, expression profiles, and cis-acting elements have been conducted in the model tree species Populus trichocarpa thus far. In this study, a comprehensive analysis of the Populus ALDH gene superfamily was performed. A total of 26 Populus ALDH genes were found to be distributed across 12 chromosomes. Genomic organization analysis indicated that purifying selection may have played a pivotal role in the retention and maintenance of PtALDH gene families. The exon-intron organizations of PtALDHs were highly conserved within the same family, suggesting that the members of the same family also may have conserved functionalities. Microarray data and qRT-PCR analysis indicated that most PtALDHs had distinct tissue-specific expression patterns. The specificity of cis-acting elements in the promoter regions of the PtALDHs and the divergence of expression patterns between nine paralogous PtALDH gene pairs suggested that gene duplications may have freed the duplicate genes from the functional constraints. The expression levels of some ALDHs were up- or down-regulated by various abiotic stresses, implying that the products of these genes may be involved in the adaptation of Populus to abiotic stresses. Overall, the data obtained from our investigation contribute to a better understanding of the complexity of the Populus ALDH gene superfamily and provide insights into the function and evolution of ALDH gene families in vascular plants.

Superfamily of G-protein coupled receptors (GPCRs – extraordinary and outstanding success of evolution

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Kazimierz Kochman

2014-10-01

Full Text Available The G protein-coupled receptors (GPCRs are considered as very diverse and also surprisingly successful structures during the whole evolutionary process, being capable of transducing the different forms of “information” within the cell and also between cells, such as different peptides, lipids, proteins, nucleotides, nucleosides, organic odorants and photons. Complex studies as well as two-dimensional crystallization of rhodopsin, their paradigm, led to the creation of a useful model having a common central core, consisting of seven transmembrane helical domains, which undergoes appropriate structural modification during activation and signal transduction. After the complete delineation of the human genome, which is the apogee of human scientific civilization and culture, it was possible to identify more than 800 human GPCR sequences and in parallel analyze 342 unique functional nonolfactory human GPCR sequences with phylogenetic analyses. These results support, with high bootstrap values, the existence of five main families, named by the authors glutamate, rhodopsin, adhesion, frizzle/taste2, and secretin, forming the GRAFS classification system. Positions of the GPCRs in chromosomal paralogous regions indicate the importance of tetraploidizations or local gene duplication events during their creation. Some families of GPCRs show, however, very little or no similarity in the sequence of amino acid chains. They utilize an enormous number of different domains to bind ligands and to activate the appropriate G-proteins. The delicate tuning of their coupling to G proteins is further regulated by splicing, RNA editing and phosphorylation. A number of GPCRs may also form homodimers or heterodimers with structurally different GPCRs and also with membrane-bound proteins having one transmembrane domain. It should also be stressed that not all GPCRs are strictly faithful to G proteins because growing evidence indicates that they can interact directly

Structural basis of transport function in major facilitator superfamily protein from Trichoderma harzianum.

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Chaudhary, Nitika; Sandhu, Padmani; Ahmed, Mushtaq; Akhter, Yusuf

2017-02-01

Trichothecenes are the sesquiterpenes secreted by Trichoderma spp. residing in the rhizosphere. These compounds have been reported to act as plant growth promoters and bio-control agents. The structural knowledge for the transporter proteins of their efflux remained limited. In this study, three-dimensional structure of Thmfs1 protein, a trichothecene transporter from Trichoderma harzianum, was homology modelled and further Molecular Dynamics (MD) simulations were used to decipher its mechanism. Fourteen transmembrane helices of Thmfs1 protein are observed contributing to an inward-open conformation. The transport channel and ligand binding sites in Thmfs1 are identified based on heuristic, iterative algorithm and structural alignment with homologous proteins. MD simulations were performed to reveal the differential structural behaviour occurring in the ligand free and ligand bound forms. We found that two discrete trichothecene binding sites are located on either side of the central transport tunnel running from the cytoplasmic side to the extracellular side across the Thmfs1 protein. Detailed analysis of the MD trajectories showed an alternative access mechanism between N and C-terminal domains contributing to its function. These results also demonstrate that the transport of trichodermin occurs via hopping mechanism in which the substrate molecule jumps from one binding site to another lining the transport tunnel. Copyright © 2016 Elsevier B.V. All rights reserved.

Extracellular Ribonuclease from Bacillus licheniformis (Balifase, a New Member of the N1/T1 RNase Superfamily

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Yulia Sokurenko

2016-01-01

Full Text Available The N1/T1 RNase superfamily comprises enzymes with well-established antitumor effects, such as ribotoxins secreted by fungi, primarily by Aspergillus and Penicillium species, and bacterial RNase secreted by B. pumilus (binase and B. amyloliquefaciens (barnase. RNase is regarded as an alternative to classical chemotherapeutic agents due to its selective cytotoxicity towards tumor cells. New RNase with a high degree of structural similarity with binase (73% and barnase (74% was isolated and purified from Bacillus licheniformis (balifase, calculated molecular weight 12421.9 Da, pI 8.91. The protein sample with enzymatic activity of 1.5 × 106 units/A280 was obtained. The physicochemical properties of balifase are similar to those of barnase. However, in terms of its gene organization and promoter activity, balifase is closer to binase. The unique feature of balifase gene organization consists in the fact that genes of RNase and its inhibitor are located in one operon. Similarly to biosynthesis of binase, balifase synthesis is induced under phosphate starvation; however, in contrast to binase, balifase does not form dimers under natural conditions. We propose that the highest stability of balifase among analyzed RNase types allows the protein to retain its structure without oligomerization.

Engineering intracellular active transport systems as in vivo biomolecular tools.

Energy Technology Data Exchange (ETDEWEB)

Bachand, George David; Carroll-Portillo, Amanda

2006-11-01

Active transport systems provide essential functions in terms of cell physiology and metastasis. These systems, however, are also co-opted by invading viruses, enabling directed transport of the virus to and from the cell's nucleus (i.e., the site of virus replication). Based on this concept, fundamentally new approaches for interrogating and manipulating the inner workings of living cells may be achievable by co-opting Nature's active transport systems as an in vivo biomolecular tool. The overall goal of this project was to investigate the ability to engineer kinesin-based transport systems for in vivo applications, specifically the collection of effector proteins (e.g., transcriptional regulators) within single cells. In the first part of this project, a chimeric fusion protein consisting of kinesin and a single chain variable fragment (scFv) of an antibody was successfully produced through a recombinant expression system. The kinesin-scFv retained both catalytic and antigenic functionality, enabling selective capture and transport of target antigens. The incorporation of a rabbit IgG-specific scFv into the kinesin established a generalized system for functionalizing kinesin with a wide range of target-selective antibodies raised in rabbits. The second objective was to develop methods of isolating the intact microtubule network from live cells as a platform for evaluating kinesin-based transport within the cytoskeletal architecture of a cell. Successful isolation of intact microtubule networks from two distinct cell types was demonstrated using glutaraldehyde and methanol fixation methods. This work provides a platform for inferring the ability of kinesin-scFv to function in vivo, and may also serve as a three-dimensional scaffold for evaluating and exploiting kinesin-based transport for nanotechnological applications. Overall, the technology developed in this project represents a first-step in engineering active transport system for in vivo

Organization of the gene coding for human protein C inhibitor (plasminogen activator inhibitor-3). Assignment of the gene to chromosome 14

NARCIS (Netherlands)

Meijers, J. C.; Chung, D. W.

1991-01-01

Protein C inhibitor (plasminogen activator inhibitor-3) is a plasma glycoprotein and a member of the serine proteinase inhibitor superfamily. In the present study, the human gene for protein C inhibitor was isolated and characterized from three independent phage that contained overlapping inserts

The Structure of a Sugar Transporter of the Glucose EIIC Superfamily Provides Insight into the Elevator Mechanism of Membrane Transport.

Science.gov (United States)

McCoy, Jason G; Ren, Zhenning; Stanevich, Vitali; Lee, Jumin; Mitra, Sharmistha; Levin, Elena J; Poget, Sebastien; Quick, Matthias; Im, Wonpil; Zhou, Ming

2016-06-07

The phosphoenolpyruvate:carbohydrate phosphotransferase systems are found in bacteria, where they play central roles in sugar uptake and regulation of cellular uptake processes. Little is known about how the membrane-embedded components (EIICs) selectively mediate the passage of carbohydrates across the membrane. Here we report the functional characterization and 2.55-Å resolution structure of a maltose transporter, bcMalT, belonging to the glucose superfamily of EIIC transporters. bcMalT crystallized in an outward-facing occluded conformation, in contrast to the structure of another glucose superfamily EIIC, bcChbC, which crystallized in an inward-facing occluded conformation. The structures differ in the position of a structurally conserved substrate-binding domain that is suggested to play a central role in sugar transport. In addition, molecular dynamics simulations suggest a potential pathway for substrate entry from the periplasm into the bcMalT substrate-binding site. These results provide a mechanistic framework for understanding substrate recognition and translocation for the glucose superfamily EIIC transporters. Copyright © 2016 Elsevier Ltd. All rights reserved.

A novel inhibitor of α9α10 nicotinic acetylcholine receptors from Conus vexillum delineates a new conotoxin superfamily.

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Sulan Luo

Full Text Available Conotoxins (CTxs selectively target a range of ion channels and receptors, making them widely used tools for probing nervous system function. Conotoxins have been previously grouped into superfamilies according to signal sequence and into families based on their cysteine framework and biological target. Here we describe the cloning and characterization of a new conotoxin, from Conus vexillum, named αB-conotoxin VxXXIVA. The peptide does not belong to any previously described conotoxin superfamily and its arrangement of Cys residues is unique among conopeptides. Moreover, in contrast to previously characterized conopeptide toxins, which are expressed initially as prepropeptide precursors with a signal sequence, a ''pro'' region, and the toxin-encoding region, the precursor sequence of αB-VxXXIVA lacks a ''pro'' region. The predicted 40-residue mature peptide, which contains four Cys, was synthesized in each of the three possible disulfide arrangements. Investigation of the mechanism of action of αB-VxXXIVA revealed that the peptide is a nicotinic acetylcholine receptor (nAChR antagonist with greatest potency against the α9α10 subtype. (1H nuclear magnetic resonance (NMR spectra indicated that all three αB-VxXXIVA isomers were poorly structured in aqueous solution. This was consistent with circular dichroism (CD results which showed that the peptides were unstructured in buffer, but adopted partially helical conformations in aqueous trifluoroethanol (TFE solution. The α9α10 nAChR is an important target for the development of analgesics and cancer chemotherapeutics, and αB-VxXXIVA represents a novel ligand with which to probe the structure and function of this protein.

Uncoupling proteins (UCP) in unicellular eukaryotes: true UCPs or UCP1-like acting proteins?

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Luévano-Martínez, Luis Alberto

2012-04-05

Uncoupling proteins belong to the superfamily of mitochondrial anion carriers. They are apparently present throughout the Eukarya domain in which only some members have an established physiological function, i.e. UCP1 from brown adipose tissue is involved in non-shivering thermogenesis. However, the proteins responsible for the phenotype observed in unicellular organisms have not been characterized. In this report we analyzed functional evidence concerning unicellular UCPs and found that true UCPs are restricted to some taxonomical groups while proteins conferring a UCP1-like phenotype to fungi and most protists are the result of a promiscuous activity exerted by other mitochondrial anion carriers. We describe a possible evolutionary route followed by these proteins by which they acquire this promiscuous mechanism. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Complement system proteins which interact with C3b or C4b A superfamily of structurally related proteins

DEFF Research Database (Denmark)

Reid, K B M; Bentley, D R; Campbell, R D

1986-01-01

Recent cDNA sequencing data has allowed the prediction of the entire amino acid sequences of complement components factor B and C2, the complement control proteins factor H and C4b-binding protein and a partial sequence for the Cab/C4b receptor CR1. These proteins all contain internal repeating u...

Glutathione Transferases Superfamily: Cold-Inducible Expression of Distinct GST Genes in Brassica oleracea

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Harshavardhanan Vijayakumar

2016-07-01

Full Text Available Plants, as sessile organisms, can suffer serious growth and developmental consequences under cold stress conditions. Glutathione transferases (GSTs, EC 2.5.1.18 are ubiquitous and multifunctional conjugating proteins, which play a major role in stress responses by preventing oxidative damage by reactive oxygen species (ROS. Currently, understanding of their function(s during different biochemical and signaling pathways under cold stress condition remain unclear. In this study, using combined computational strategy, we identified 65 Brassica oleracea glutathione transferases (BoGST and characterized them based on evolutionary analysis into 11 classes. Inter-species and intra-species duplication was evident between BoGSTs and Arabidopsis GSTs. Based on localization analyses, we propose possible pathways in which GST genes are involved during cold stress. Further, expression analysis of the predicted putative functions for GST genes were investigated in two cold contrasting genotypes (cold tolerance and susceptible under cold condition, most of these genes were highly expressed at 6 h and 1 h in the cold tolerant (CT and cold susceptible (CS lines, respectively. Overall, BoGSTU19, BoGSTU24, BoGSTF10 are candidate genes highly expressed in B. oleracea. Further investigation of GST superfamily in B. oleracea will aid in understanding complex mechanism underlying cold tolerance in plants.

Structural mutations of C-domains in members of the Ig superfamily. Consequences for the interactions between the T cell antigen receptor and the zeta 2 homodimer

DEFF Research Database (Denmark)

Geisler, C; Rubin, B; Caspar-Bauguil, S

1992-01-01

Several molecules belonging to the Ig superfamily are expressed together with noncovalently associated subunits. This applies for membrane-bound IgM and IgD, some of the FcR, and the Ti dimers of the TCR. The interactions between members of the Ig superfamily and their associated subunits are sti...

Microdomain forming proteins in oncogenesis

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I. B. Zborovskaya

2016-01-01

Full Text Available Lipid rafts are lateral assembles of cholesterol, sphingomyelin, glicosphingolipids and specific proteins within cell plasma membrane. These microdomains are involved into a number of important cellular processes including membrane rearrangement, protein internalization, signal transduction, entry of viruses into the cell. Some of lipid rafts are stabilized by special microdomain-forming proteins such as caveolins, SPFH domain containing superfamily, tetraspanins, galectins, which maintain integrity of rafts and regulate signal transduction via forming of “signalosomes”. Involvement of the different lipid rafts is necessary in many situations such as binding of growth factors with their receptors, integrin regulation, cytoskeleton and extracellular matrix rearrangements, vesicular transport, etc. However, such classes of microdomain-forming proteins are still considered separately from each other. In this review we tried to perform complex analysis of microdomain-forming proteins in regulation of cancer assotiated processes.

Production and characterization of a thermostable alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily

NARCIS (Netherlands)

Machielsen, M.P.; Uria, A.R.; Kengen, S.W.M.; Oost, van der J.

2006-01-01

The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The

Targeted deletion of Kif18a protects from colitis-associated colorectal (CAC) tumors in mice through impairing Akt phosphorylation

International Nuclear Information System (INIS)

Zhu, Houbao; Xu, Wangyang; Zhang, Hongxin; Liu, Jianbing; Xu, Haimin; Lu, Shunyuan; Dang, Suying; Kuang, Ying; Jin, Xiaolong; Wang, Zhugang

2013-01-01

Highlights: •Kif18A is up-regulated in CAC of mouse model. •Kif18a −/− mice are protected from CAC. •Tumor cells from Kif18a −/− mice undergo more apoptosis. •Kif18A deficiency induces poor Atk phosphorylation. -- Abstract: Kinesins are a superfamily of molecular motors involved in cell division or intracellular transport. They are becoming important targets for chemotherapeutic intervention of cancer due to their crucial role in mitosis. Here, we demonstrate that the kinesin-8 Kif18a is overexpressed in murine CAC and is a crucial promoter during early CAC carcinogenesis. Kif18a-deficient mice are evidently protected from AOM–DSS-induced colon carcinogenesis. Kif18A is responsible for proliferation of colonic tumor cells, while Kif18a ablation in mice promotes cell apoptosis. Mechanistically, Kif18a is responsible for induction of Akt phosphorylation, which is known to be associated with cell survival regulation. In conclusion, Kif18a is critical for colorectal carcinogenesis in the setting of inflammation by mechanisms of increased PI3K-AKT signaling. Inhibition of Kif18A activity may be useful in the prevention or chemotherapeutic intervention of CAC

Evolution of Enzymatic Activities in the Enolase Superfamily: Stereochemically Distinct Mechanisms in Two Families of cis,cis-Muconate Lactonizing Enzymes

Energy Technology Data Exchange (ETDEWEB)

Sakai, A.; Fedorov, A; Fedorov, E; Schnoes, A; Glasner, M; Burley, S; Babbitt, P; Almo, S; Gerlt, J

2009-01-01

The mechanistically diverse enolase superfamily is a paradigm for elucidating Nature's strategies for divergent evolution of enzyme function. Each of the different reactions catalyzed by members of the superfamily is initiated by abstraction of the a-proton of a carboxylate substrate that is coordinated to an essential Mg2+. The muconate lactonizing enzyme (MLE) from Pseudomonas putida, a member of a family that catalyzes the syn-cycloisomerization of cis,cis-muconate to (4S)-muconolactone in the e-ketoadipate pathway, has provided critical insights into the structural bases for evolution of function within the superfamily. A second, divergent family of homologous MLEs that catalyzes anti-cycloisomerization has been identified. Structures of members of both families liganded with the common (4S)-muconolactone product (syn, Pseudomonas fluorescens, gi 70731221; anti, Mycobacterium smegmatis, gi 118470554) document that the conserved Lys at the end of the second e-strand in the (e/a)7e-barrel domain serves as the acid catalyst in both reactions. The different stereochemical courses (syn and anti) result from different structural strategies for determining substrate specificity: although the distal carboxylate group of the cis,cis-muconate substrate attacks the same face of the proximal double bond, opposite faces of the resulting enolate anion intermediate are presented to the conserved Lys acid catalyst. The discovery of two families of homologous, but stereochemically distinct, MLEs likely provides an example of 'pseudoconvergent' evolution of the same function from different homologous progenitors within the enolase superfamily, in which different spatial arrangements of active site functional groups and substrate specificity determinants support catalysis of the same reaction.

Evolution of Enzymatic Activities in the Enolase Superfamily: Stereochemically Distinct Mechanisms in Two Families of cis,cis-Muconate Lactonizing Enzymes†

Science.gov (United States)

Sakai, Ayano; Fedorov, Alexander A.; Fedorov, Elena V.; Schnoes, Alexandra M.; Glasner, Margaret E.; Brown, Shoshana; Rutter, Marc E.; Bain, Kevin; Chang, Shawn; Gheyi, Tarun; Sauder, J. Michael; Burley, Stephen K.; Babbitt, Patricia C.; Almo, Steven C.; Gerlt, John A.

2009-01-01

The mechanistically diverse enolase superfamily is a paradigm for elucidating Nature’s strategies for divergent evolution of enzyme function. Each of the different reactions catalyzed by members of the superfamily is initiated by abstraction of the α-proton of a carboxylate substrate that is coordinated to an essential Mg2+. The muconate lactonizing enzyme (MLE) from Pseudomonas putida, a member of a family that catalyzes the syn-cycloisomerization of cis,cis-muconate to (4S)-muconolactone in the β-ketoadipate pathway, has provided critical insights into the structural bases for evolution of function within the superfamily. A second, divergent family of homologues MLEs that catalyzes anti-cycloisomerization has been identified. Structures of members of both families liganded with the common (4S)-muconolactone product (syn, Pseudomonas fluorescens, GI:70731221; anti, Mycobacterium smegmatis, GI:118470554) document that the conserved Lys at the end of the second β-strand in the (β/α)7β-barrel domain serves as the acid catalyst in both reactions. The different stereochemical courses (syn and anti) result from different structural strategies for determining substrate specificity: although the distal carboxylate group of the cis,cis-muconate substrate attacks the same face of the proximal double bond, opposite faces of the resulting enolate anion intermediate are presented to the conserved Lys acid catalyst. The discovery of two families of homologous, but stereochemically distinct, MLEs likely provides an example of “pseudoconvergent” evolution of the same function from different homologous progenitors within the enolase superfamily, in which different spatial arrangements of active site functional groups and substrate specificity determinants support catalysis of the same reaction. PMID:19220063

G Protein-Coupled Receptor Signaling in Stem Cells and Cancer.

Science.gov (United States)

Lynch, Jennifer R; Wang, Jenny Yingzi

2016-05-11

G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

G Protein-Coupled Receptor Signaling in Stem Cells and Cancer

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Jennifer R. Lynch

2016-05-01

Full Text Available G protein-coupled receptors (GPCRs are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84 and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

Phylogenetic analysis and protein structure modelling identifies distinct Ca(2+)/Cation antiporters and conservation of gene family structure within Arabidopsis and rice species.

Science.gov (United States)

Pittman, Jon K; Hirschi, Kendal D

2016-12-01

The Ca(2+)/Cation Antiporter (CaCA) superfamily is an ancient and widespread family of ion-coupled cation transporters found in nearly all kingdoms of life. In animals, K(+)-dependent and K(+)-indendent Na(+)/Ca(2+) exchangers (NCKX and NCX) are important CaCA members. Recently it was proposed that all rice and Arabidopsis CaCA proteins should be classified as NCX proteins. Here we performed phylogenetic analysis of CaCA genes and protein structure homology modelling to further characterise members of this transporter superfamily. Phylogenetic analysis of rice and Arabidopsis CaCAs in comparison with selected CaCA members from non-plant species demonstrated that these genes form clearly distinct families, with the H(+)/Cation exchanger (CAX) and cation/Ca(2+) exchanger (CCX) families dominant in higher plants but the NCKX and NCX families absent. NCX-related Mg(2+)/H(+) exchanger (MHX) and CAX-related Na(+)/Ca(2+) exchanger-like (NCL) proteins are instead present. Analysis of genomes of ten closely-related rice species and four Arabidopsis-related species found that CaCA gene family structures are highly conserved within related plants, apart from minor variation. Protein structures were modelled for OsCAX1a and OsMHX1. Despite exhibiting broad structural conservation, there are clear structural differences observed between the different CaCA types. Members of the CaCA superfamily form clearly distinct families with different phylogenetic, structural and functional characteristics, and therefore should not be simply classified as NCX proteins, which should remain as a separate gene family.

Evolutionary distance from human homologs reflects allergenicity of animal food proteins.

Science.gov (United States)

Jenkins, John A; Breiteneder, Heimo; Mills, E N Clare

2007-12-01

In silico analysis of allergens can identify putative relationships among protein sequence, structure, and allergenic properties. Such systematic analysis reveals that most plant food allergens belong to a restricted number of protein superfamilies, with pollen allergens behaving similarly. We have investigated the structural relationships of animal food allergens and their evolutionary relatedness to human homologs to define how closely a protein must resemble a human counterpart to lose its allergenic potential. Profile-based sequence homology methods were used to classify animal food allergens into Pfam families, and in silico analyses of their evolutionary and structural relationships were performed. Animal food allergens could be classified into 3 main families--tropomyosins, EF-hand proteins, and caseins--along with 14 minor families each composed of 1 to 3 allergens. The evolutionary relationships of each of these allergen superfamilies showed that in general, proteins with a sequence identity to a human homolog above approximately 62% were rarely allergenic. Single substitutions in otherwise highly conserved regions containing IgE epitopes in EF-hand parvalbumins may modulate allergenicity. These data support the premise that certain protein structures are more allergenic than others. Contrasting with plant food allergens, animal allergens, such as the highly conserved tropomyosins, challenge the capability of the human immune system to discriminate between foreign and self-proteins. Such immune responses run close to becoming autoimmune responses. Exploiting the closeness between animal allergens and their human homologs in the development of recombinant allergens for immunotherapy will need to consider the potential for developing unanticipated autoimmune responses.

The viral transmembrane superfamily: possible divergence of Arenavirus and Filovirus glycoproteins from a common RNA virus ancestor

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Buchmeier Michael J

2001-02-01

Full Text Available Abstract Background Recent studies of viral entry proteins from influenza, measles, human immunodeficiency virus, type 1 (HIV-1, and Ebola virus have shown, first with molecular modeling, and then X-ray crystallographic or other biophysical studies, that these disparate viruses share a coiled-coil type of entry protein. Results Structural models of the transmembrane glycoproteins (GP-2 of the Arenaviruses, lymphochoriomeningitis virus (LCMV and Lassa fever virus, are presented, based on consistent structural propensities despite variation in the amino acid sequence. The principal features of the model, a hydrophobic amino terminus, and two antiparallel helices separated by a glycosylated, antigenic apex, are common to a number of otherwise disparate families of enveloped RNA viruses. Within the first amphipathic helix, demonstrable by circular dichroism of a peptide fragment, there is a highly conserved heptad repeat pattern proposed to mediate multimerization by coiled-coil interactions. The amino terminal 18 amino acids are 28% identical and 50% highly similar to the corresponding region of Ebola, a member of the Filovirus family. Within the second, charged helix just prior to membrane insertion there is also high similarity over the central 18 amino acids in corresponding regions of Lassa and Ebola, which may be further related to the similar region of HIV-1 defining a potent antiviral peptide analogue. Conclusions These findings indicate a common pattern of structure and function among viral transmembrane fusion proteins from a number of virus families. Such a pattern may define a viral transmembrane superfamily that evolved from a common precursor eons ago.

Klp10A modulates the localization of centriole-associated proteins during Drosophila male gametogenesis.

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Gottardo, Marco; Callaini, Giuliano; Riparbelli, Maria Giovanna

2016-12-16

Mutations in Klp10A, a microtubule-depolymerising Kinesin-13, lead to overly long centrioles in Drosophila male germ cells. We demonstrated that the loss of Klp10A modifies the distribution of typical proteins involved in centriole assembly and function. In the absence of Klp10A the distribution of Drosophila pericentrin-like protein (Dplp), Sas-4 and Sak/Plk4 that are restricted in control testes to the proximal end of the centriole increase along the centriole length. Remarkably, the cartwheel is lacking or it appears abnormal in mutant centrioles, suggesting that this structure may spatially delimit protein localization. Moreover, the parent centrioles that in control cells have the same dimensions grow at different rates in mutant testes with the mother centrioles longer than the daughters. Daughter centrioles have often an ectopic position with respect to the proximal end of the mothers and failed to recruit Dplp.

Building a Phylogenetic Tree of the Human and Ape Superfamily Using DNA-DNA Hybridization Data

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Maier, Caroline Alexander

2004-01-01

The study describes the process of DNA-DNA hybridization and the history of its use by Sibley and Alquist in simple, straightforward, and interesting language that students easily understand to create their own phylogenetic tree of the hominoid superfamily. They calibrate the DNA clock and use it to estimate the divergence dates of the various…

ARCPHdb: A comprehensive protein database for SF1 and SF2 helicase from archaea.

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Moukhtar, Mirna; Chaar, Wafi; Abdel-Razzak, Ziad; Khalil, Mohamad; Taha, Samir; Chamieh, Hala

2017-01-01

Superfamily 1 and Superfamily 2 helicases, two of the largest helicase protein families, play vital roles in many biological processes including replication, transcription and translation. Study of helicase proteins in the model microorganisms of archaea have largely contributed to the understanding of their function, architecture and assembly. Based on a large phylogenomics approach, we have identified and classified all SF1 and SF2 protein families in ninety five sequenced archaea genomes. Here we developed an online webserver linked to a specialized protein database named ARCPHdb to provide access for SF1 and SF2 helicase families from archaea. ARCPHdb was implemented using MySQL relational database. Web interfaces were developed using Netbeans. Data were stored according to UniProt accession numbers, NCBI Ref Seq ID, PDB IDs and Entrez Databases. A user-friendly interactive web interface has been developed to browse, search and download archaeal helicase protein sequences, their available 3D structure models, and related documentation available in the literature provided by ARCPHdb. The database provides direct links to matching external databases. The ARCPHdb is the first online database to compile all protein information on SF1 and SF2 helicase from archaea in one platform. This database provides essential resource information for all researchers interested in the field. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fundamental Characteristics of AAA+ Protein Family Structure and Function.

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Miller, Justin M; Enemark, Eric J

2016-01-01

Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins.

The Myriad Roles of Miro in the Nervous System: Axonal Transport of Mitochondria and Beyond

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Bingwei eLu

2014-10-01

Full Text Available Mitochondrial rho GTPase (Miro is a mitochondrial outer membrane protein containing two GTPase domains and two helix-loop-helix Ca2+-binding domains called EF hands. Pioneering genetic studies in Drosophila first revealed a key function of Miro in regulating the axonal transport of mitochondria, during which Miro forms a multi-protein transport complex with Milton and Kinesin heavy chain (KHC to link trafficking mitochondria with the microtubule cytoskeleton. Recent studies showed that through binding to the EF hands of Miro and causing conformational changes of Miro and alteration of protein-protein interactions within the transport complex, Ca2+ can alter the engagement of mitochondria with the microtubule (MT/kinesin network, offering one mechanism to match mitochondrial distribution with neuronal activity. Despite the importance of the Miro/Milton/Kinesin complex in regulating mitochondrial transport in metazoans, not all components of the transport complex are conserved in lower organisms, and transport-independent functions of Miro are emerging. Here we review the diverse functions of the evolutionarily conserved Miro proteins that are relevant to the development, maintenance, and functioning of the nervous system and discuss the potential contribution of Miro dysfunction to the pathogenesis of diseases of the nervous system.

Gclust Server: 94819 [Gclust Server

Lifescience Database Archive (English)

Full Text Available ted protein required for both karyogamy and mitotic spindle organization, interacts stably and specifically ...ve annotation Kinesin-associated protein required for both karyogamy and mitotic spindle organization, inter

Expression of TGF-beta superfamily growth factors, their receptors, the associated SMADs and antagonists in five isolated size-matched populations of pre-antral follicles from normal human ovaries

DEFF Research Database (Denmark)

Kristensen, Stine Gry; Andersen, Kasper; Clement, Christian Alexandro

2014-01-01

In mammals, members of the transforming growth factor-beta (TGF-β) superfamily are known to have key roles in the regulation of follicular growth and development. The aim of the study was to evaluate the expression of TGF-β superfamily growth factors, their receptors, downstream SMAD signalling m...... growth. Moreover, the presence of multiple TGF-β/BMP antagonists imply that certain growth factors are subjected to local regulation on different levels which address another important level of intraovarian regulation of follicle development in humans.......In mammals, members of the transforming growth factor-beta (TGF-β) superfamily are known to have key roles in the regulation of follicular growth and development. The aim of the study was to evaluate the expression of TGF-β superfamily growth factors, their receptors, downstream SMAD signalling...... molecules and TGF- β/BMP antagonists during early human folliculogenesis.Human preantral follicles were enzymatically isolated from surplus ovarian tissue obtained from women having ovarian cortical tissue frozen for fertility preservation. A total of 348 human preantral follicles, ranging from 40 to 200 µm...

Bone morphogenetic proteins regulate osteoprotegerin and its ligands in human vascular smooth muscle cells

DEFF Research Database (Denmark)

Knudsen, Kirsten Quyen Nguyen; Olesen, Ping; Ledet, Thomas

2007-01-01

The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved in the transformat......The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved...... in the transformation of human vascular smooth muscle cells (HVSMC) to osteoblast-like cells. In this study, we evaluated the effect of BMP-2, BMP-7 and transforming growth factor beta (TGF-beta1) on the secretion and mRNA expression of OPG and its ligands receptor activator of nuclear factor-kappabeta ligand (RANKL......) and TNF-related apoptosis-inducing ligand (TRAIL) in HVSMC. All three growth factors decreased OPG protein production significantly; these results were paralleled by reduced OPG mRNA expression. TRAIL mRNA levels were also decreased. RANKL mRNA expression declined when treated with TGF-beta1 but were...

Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): Identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42

International Nuclear Information System (INIS)

Shinjo, K.; Koland, J.G.; Hart, M.J.; Narasimhan, V.; Cerione, R.A.; Johnson, D.I.; Evans, T.

1990-01-01

The authors have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated G p (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human G p protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins and the rac proteins. The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell division-cycle protein CDC42. The human placental gene, which they designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein

The evolution of function in strictosidine synthase-like proteins.

Science.gov (United States)

Hicks, Michael A; Barber, Alan E; Giddings, Lesley-Ann; Caldwell, Jenna; O'Connor, Sarah E; Babbitt, Patricia C

2011-11-01

The exponential growth of sequence data provides abundant information for the discovery of new enzyme reactions. Correctly annotating the functions of highly diverse proteins can be difficult, however, hindering use of this information. Global analysis of large superfamilies of related proteins is a powerful strategy for understanding the evolution of reactions by identifying catalytic commonalities and differences in reaction and substrate specificity, even when only a few members have been biochemically or structurally characterized. A comparison of >2500 sequences sharing the six-bladed β-propeller fold establishes sequence, structural, and functional links among the three subgroups of the functionally diverse N6P superfamily: the arylesterase-like and senescence marker protein-30/gluconolactonase/luciferin-regenerating enzyme-like (SGL) subgroups, representing enzymes that catalyze lactonase and related hydrolytic reactions, and the so-called strictosidine synthase-like (SSL) subgroup. Metal-coordinating residues were identified as broadly conserved in the active sites of all three subgroups except for a few proteins from the SSL subgroup, which have been experimentally determined to catalyze the quite different strictosidine synthase (SS) reaction, a metal-independent condensation reaction. Despite these differences, comparison of conserved catalytic features of the arylesterase-like and SGL enzymes with the SSs identified similar structural and mechanistic attributes between the hydrolytic reactions catalyzed by the former and the condensation reaction catalyzed by SS. The results also suggest that despite their annotations, the great majority of these >500 SSL sequences do not catalyze the SS reaction; rather, they likely catalyze hydrolytic reactions typical of the other two subgroups instead. This prediction was confirmed experimentally for one of these proteins. Copyright © 2011 Wiley-Liss, Inc.

Anatomo-pathological aspects of parasitism by nematodes of the superfamily Metastrongyloidea in wild crab-eating fox (Cerdocyon thous in Midwestern Brazil

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Jair Alves Ferreira Júnior

Full Text Available ABSTRACT: Nematodes of the superfamily Metastrongyloidea affect the respiratory, cardiovascular, and nervous systems of domestic carnivores and are uncommonly detected in wild animals. This report describes the lesions associated with pulmonary parasitism by nematodes of the superfamily Metastrongyloidea in a wild crab-eating fox ( Cerdocyon thous in the Federal District, Brazil. Grossly, there was pulmonary hyperemia, edema, and emphysema. Microscopically, there was granulomatous arteritis associated with intravascular metastrongylid. The anatomical location, characteristic lesion, and histological features of the parasite suggested that the nematode involved in this case is Angiostrongylus vasorum . This worm is frequently reported parasitizing pulmonary arteries of domestic canids but is uncommonly described in wild canids in Midwestern Brazil.

Deleted in malignant brain tumors-1 protein (DMBT1): a pattern recognition receptor with multiple binding sites.

Science.gov (United States)

Ligtenberg, Antoon J M; Karlsson, Niclas G; Veerman, Enno C I

2010-01-01

Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1(SAG)), and lung glycoprotein-340 (DMBT1(GP340)) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW). Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.

Deleted in Malignant Brain Tumors-1 Protein (DMBT1: A Pattern Recognition Receptor with Multiple Binding Sites

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Enno C. I. Veerman

2010-12-01

Full Text Available Deleted in Malignant Brain Tumors-1 protein (DMBT1, salivary agglutinin (DMBT1SAG, and lung glycoprotein-340 (DMBT1GP340 are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW. Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.

[Cloning, mutagenesis and symbiotic phenotype of three lipid transfer protein encoding genes from Mesorhizobium huakuii 7653R].

Science.gov (United States)

Li, Yanan; Zeng, Xiaobo; Zhou, Xuejuan; Li, Youguo

2016-12-04

Lipid transfer protein superfamily is involved in lipid transport and metabolism. This study aimed to construct mutants of three lipid transfer protein encoding genes in Mesorhizobium huakuii 7653R, and to study the phenotypes and function of mutations during symbiosis with Astragalus sinicus. We used bioinformatics to predict structure characteristics and biological functions of lipid transfer proteins, and conducted semi-quantitative and fluorescent quantitative real-time PCR to analyze the expression levels of target genes in free-living and symbiotic conditions. Using pK19mob insertion mutagenesis to construct mutants, we carried out pot plant experiments to observe symbiotic phenotypes. MCHK-5577, MCHK-2172 and MCHK-2779 genes encoding proteins belonged to START/RHO alpha_C/PITP/Bet_v1/CoxG/CalC (SRPBCC) superfamily, involved in lipid transport or metabolism, and were identical to M. loti at 95% level. Gene relative transcription level of the three genes all increased compared to free-living condition. We obtained three mutants. Compared with wild-type 7653R, above-ground biomass of plants and nodulenitrogenase activity induced by the three mutants significantly decreased. Results indicated that lipid transfer protein encoding genes of Mesorhizobium huakuii 7653R may play important roles in symbiotic nitrogen fixation, and the mutations significantly affected the symbiotic phenotypes. The present work provided a basis to study further symbiotic function mechanism associated with lipid transfer proteins from rhizobia.

Targeted deletion of Kif18a protects from colitis-associated colorectal (CAC) tumors in mice through impairing Akt phosphorylation

Energy Technology Data Exchange (ETDEWEB)

Zhu, Houbao [State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui-Jin Hospital and Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025 (China); Xu, Wangyang [Department of Clinical Laboratories, Ninth People’s Hospital, SJTUSM, Shanghai 200011 (China); Zhang, Hongxin [State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui-Jin Hospital and Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025 (China); Liu, Jianbing [State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui-Jin Hospital and Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025 (China); Shanghai Research Center for Model Organisms, Shanghai 201203 (China); Xu, Haimin [Department of Pathology, Rui-Jin Hospital, SJTUSM, Shanghai 200025 (China); Lu, Shunyuan; Dang, Suying [State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui-Jin Hospital and Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025 (China); Kuang, Ying [Shanghai Research Center for Model Organisms, Shanghai 201203 (China); Jin, Xiaolong [Department of Pathology, Rui-Jin Hospital, SJTUSM, Shanghai 200025 (China); Wang, Zhugang, E-mail: zhugangw@shsmu.edu.cn [State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui-Jin Hospital and Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025 (China); Shanghai Research Center for Model Organisms, Shanghai 201203 (China)

2013-08-16

Highlights: •Kif18A is up-regulated in CAC of mouse model. •Kif18a{sup −/−} mice are protected from CAC. •Tumor cells from Kif18a{sup −/−} mice undergo more apoptosis. •Kif18A deficiency induces poor Atk phosphorylation. -- Abstract: Kinesins are a superfamily of molecular motors involved in cell division or intracellular transport. They are becoming important targets for chemotherapeutic intervention of cancer due to their crucial role in mitosis. Here, we demonstrate that the kinesin-8 Kif18a is overexpressed in murine CAC and is a crucial promoter during early CAC carcinogenesis. Kif18a-deficient mice are evidently protected from AOM–DSS-induced colon carcinogenesis. Kif18A is responsible for proliferation of colonic tumor cells, while Kif18a ablation in mice promotes cell apoptosis. Mechanistically, Kif18a is responsible for induction of Akt phosphorylation, which is known to be associated with cell survival regulation. In conclusion, Kif18a is critical for colorectal carcinogenesis in the setting of inflammation by mechanisms of increased PI3K-AKT signaling. Inhibition of Kif18A activity may be useful in the prevention or chemotherapeutic intervention of CAC.

In vivo control mechanisms of motor-cargo movement on microtubules

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Gunawardena, Shermali

2014-03-01

Within axons, molecular motors transport essential components required for neuronal growth and viability. Although many levels of regulation must exist for proper anterograde and retrograde transport of vital proteins, little is known about these mechanisms. Previous work suggested that the amyloid precursor protein (APP) functions as a kinesin-1 receptor during transport. However, how APP vesicle motility is regulated is unclear. Using genetics and in vivo imaging in Drosophila we showed that reduction of presenilin (PS) substantially increased anterograde and retrograde APP vesicle velocities. Strikingly, PS deficiency had no effect on an unrelated cargo vesicle containing synaptotagmin, which is powered by a different kinesin motor. Increased PS-mediated velocities required functional kinesin-1 and dynein motors. We also found that these PS-mediated effects on motor protein function were mediated via a pathway that involves glycogen synthase kinase-3 β (GSK-3 β) . PS genetically interacted with GSK-3 β in an activity dependent manner. Excess of active GSK-3 β perturbed transport by causing axonal blockages, which were enhanced by reduction of kinesin-1 or dynein, while excess of non-functional GSK-3 β had no effect. Strikingly, GSK-3 β-activity dependent transport defects were enhanced by reduction of PS. Collectively, our findings suggest that PS and GSK-3 β are required for normal motor protein function, and we propose a model in which PS likely regulates GSK-3 β activity during transport. These findings have important implications for our understanding of the complex regulatory machinery that must exist in vivo and how this system is coordinated during vesicle motility on microtubules.

Understanding the -C-X1-X2-C- motif in the active site of the thioredoxin superfamily: E. coli DsbA and its mutants as a model system.

Science.gov (United States)

Karshikoff, Andrey; Nilsson, Lennart; Foloppe, Nicolas

2013-08-27

E. coli DsbA is an intensively studied enzyme of the thioredoxin superfamily of thiol-disulfide oxidoreductases. DsbA catalyzes the disulfide bond formation and folding of proteins in the bacterial periplasm. DsbA and its mutants have highlighted the strong and puzzling influence of the -C-X1-X2-C- active site variants, found across the thioredoxin superfamily, on the ionization and redox properties of this site. However, the interpretation of these observations remains wanting, largely due to a dearth of structural information. Here, molecular dynamics simulations are used to provide extensive information on the structure and dynamics of reduced -C30-X31-X32-C33- motifs in wild type DsbA and 13 of its mutants. These simulations are combined with calculations of the pK of H32 and of the very low pK of the catalytic cysteine C30. In wild type DsbA, the titrations of C30 and H32 are shown to be coupled; the protonation states and dynamics of H32 are examined. The thiolate of C30 is stabilized by hydrogen bonds with the protein. Modulation of these hydrogen bonds by alteration of residue X32 has the greatest impact on the pK of C30, which rationalizes its higher pK in thioredoxin and tryparedoxin. Because of structural constrains, residue X31 has only an indirect and weak influence on the pK of C30. The dynamics of C30 is clearly related to its stabilizing interactions and pK value. Although relatively small differences between pKs were not reproduced in the calculations, the major trends are explained, adding new insights to our understanding of enzymes in this family.

Characterization of two bacterial hydroxynitrile lyases with high similarity to cupin superfamily proteins

NARCIS (Netherlands)

Hussain, Z.; Wiedner, R.; Steiner, K.; Hajek, T.; Avi, M.; Hecher, B.; Sessitsch, A.; Schwab, H.

2012-01-01

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins. In the reverse reaction, they catalyze the formation of carbon-carbon bonds by enantioselective condensation of hydrocyanic acid with carbonyls. In this study, we describe two proteins from endophytic bacteria that display activity

Role of a new member of IGFBP superfamily, IGFBP-rP10, in proliferation and differentiation of osteoblastic cells

International Nuclear Information System (INIS)

Shibata, Yasuaki; Tsukazaki, Tomoo; Hirata, Kazunari; Xin Cheng; Yamaguchi, Akira

2004-01-01

Bone regeneration is critically regulated by various molecules. To identify the new genes involved in bone regeneration, we performed microarray-based gene expression analysis using a mouse bone regeneration model. We identified a new member of the IGFBP superfamily, designated IGFBP-rP10, whose expression is up-regulated at the early phase of bone regeneration. IGFBP-rP10 consists of an IGFBP homologous domain followed by a Kazal-type protein inhibitor domain and an immunoglobulin G-like domain. A real-time-based RT-PCR analysis demonstrated that various tissues including bone expressed IGFBP-rP10 mRNA in various degrees, and confirmed an up-regulation at the early phase of bone regeneration. In situ hybridization revealed that osteoblastic cells expressed IGFPB-rP10 mRNA during bone regeneration. Bone morphogenetic protein-2 increased the expression level of IGFBP-rP10 mRNA in various cells including C3H10T1/2, MC3T3-E1, C2C12, and primary murine osteoblastic cells. The addition of recombinant mouse IGFBP-rP10 promoted the proliferation of these cells but failed to stimulate alkaline phosphatase activity. These results suggest that IGFBP-rP10 is involved in the proliferation of osteoblasts during bone formation and bone regeneration

F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.

Science.gov (United States)

Liu, Suxuan; Xiong, Xinyu; Zhao, Xianxian; Yang, Xiaofeng; Wang, Hong

2015-05-09

Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting

Genetic polymorphisms of tumour necrosis factor receptor superfamily 1b and fas ligand are associated with clinical efficacy and/or acute severe infusion reactions to infliximab in Crohn's disease

DEFF Research Database (Denmark)

Steenholdt, C; Enevold, C; Ainsworth, M A

2012-01-01

Single nucleotide polymorphisms (SNPs) in TNF receptor superfamily (TNFRSF) 1A and 1B, and Fas ligand (FASLG) genes, have been associated with responsiveness to infliximab (IFX) in Crohn's disease.......Single nucleotide polymorphisms (SNPs) in TNF receptor superfamily (TNFRSF) 1A and 1B, and Fas ligand (FASLG) genes, have been associated with responsiveness to infliximab (IFX) in Crohn's disease....

The CDM Superfamily Protein MBC Directs Myoblast Fusion through a Mechanism That Requires Phosphatidylinositol 3,4,5-Triphosphate Binding but Is Independent of Direct Interaction with DCrk▿§

Science.gov (United States)

Balagopalan, Lakshmi; Chen, Mei-Hui; Geisbrecht, Erika R.; Abmayr, Susan M.

2006-01-01

myoblast city (mbc), a member of the CDM superfamily, is essential in the Drosophila melanogaster embryo for fusion of myoblasts into multinucleate fibers. Using germ line clones in which both maternal and zygotic contributions were eliminated and rescue of the zygotic loss-of-function phenotype, we established that mbc is required in the fusion-competent subset of myoblasts. Along with its close orthologs Dock180 and CED-5, MBC has an SH3 domain at its N terminus, conserved internal domains termed DHR1 and DHR2 (or “Docker”), and C-terminal proline-rich domains that associate with the adapter protein DCrk. The importance of these domains has been evaluated by the ability of MBC mutations and deletions to rescue the mbc loss-of-function muscle phenotype. We demonstrate that the SH3 and Docker domains are essential. Moreover, ethyl methanesulfonate-induced mutations that change amino acids within the MBC Docker domain to residues that are conserved in other CDM family members nevertheless eliminate MBC function in the embryo, which suggests that these sites may mediate interactions specific to Drosophila MBC. A functional requirement for the conserved DHR1 domain, which binds to phosphatidylinositol 3,4,5-triphosphate, implicates phosphoinositide signaling in myoblast fusion. Finally, the proline-rich C-terminal sites mediate strong interactions with DCrk, as expected. These sites are not required for MBC to rescue the muscle loss-of-function phenotype, however, which suggests that MBC's role in myoblast fusion can be carried out independently of direct DCrk binding. PMID:17030600

Update of the human secretoglobin (SCGB gene superfamily and an example of 'evolutionary bloom' of androgen-binding protein genes within the mouse Scgb gene superfamily

Directory of Open Access Journals (Sweden)

Jackson Brian C

2011-10-01

Full Text Available Abstract The secretoglobins (SCGBs comprise a family of small, secreted proteins found in animals exclusively of mammalian lineage. There are 11 human SCGB genes and five pseudogenes. Interestingly, mice have 68 Scgb genes, four of which are highly orthologous to human SCGB genes; the remainder represent an 'evolutionary bloom' and make up a large gene family represented by only six counterparts in humans. SCGBs are found in high concentrations in many mammalian secretions, including fluids of the lung, lacrimal gland, salivary gland, prostate and uterus. Whereas the biological activities of most individual SCGBs have not been fully characterised, what already has been discovered suggests that this family has an important role in the modulation of inflammation, tissue repair and tumorigenesis. In mice, the large Scgb1b and Scgb2b gene families encode the androgen-binding proteins, which have been shown to play a role in mate selection. Although much has been learned about SCGBs in recent years, clearly more research remains to be done to allow a better understanding of the roles of these proteins in human health and disease. Such information is predicted to reveal valuable novel drug targets for the treatment of inflammation, as well as designing biomarkers that might identify tissue damage or cancer.

Quantitative phosphoproteomic analysis of early alterations in protein phosphorylation by 2,3,7,8-tetrachlorodibenzo-p-dioxin

DEFF Research Database (Denmark)

Schulz, Melanie; Brandner, Stefanie; Eberhagen, Carola

2013-01-01

A comprehensive quantitative analysis of changes in protein phosphorylation preceding or accompanying transcriptional activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 5L rat hepatoma cells was performed using the SILAC approach. Following exposure of the cells to DMSO or 1 nM TCDD for 0......-induced gene activation, regulators of small GTPases of the Ras superfamily, UBX domain-containing proteins and the oncogenic protein LYRIC. The results open up new directions for research on the molecular mechanisms of dioxin action and toxicity....

Galatheoidea are not monophyletic - molecular and morphological phylogeny of the squat lobsters (Decapoda: Anomura) with recognition of a new superfamily.

Science.gov (United States)

Schnabel, K E; Ahyong, S T; Maas, E W

2011-02-01

The monophyletic status of the squat lobster superfamily Galatheoidea has come under increasing doubt by studies using evidence as diverse as larval and adult somatic morphology, sperm ultrastructure, and molecular data. Here we synthesize phylogenetic data from these diverse strands, with the addition of new molecular and morphological data to examine the phylogeny of the squat lobsters and assess the status of the Galatheoidea. A total of 64 species from 16 of the 17 currently recognised anomuran families are included. Results support previous work pointing towards polyphyly in the superfamily Galatheoidea and Paguroidea, specifically, suggesting independent origins of the Galatheidae+Porcellanidae and the Chirostylidae+Kiwaidae. Morphological characters are selected that support clades resolved in the combined analysis and the taxonomic status of Galatheoidea sensu lato is revised. Results indicate that Chirostylidae are more closely related to an assemblage including Aegloidea, Lomisoidea and Paguroidea than to the remaining Galatheoidea and are referred to the superfamily Chirostyloidea to include the Chirostylidae and Kiwaidae. A considerable amount of research highlighting morphological differences supporting this split is discussed. The Galatheoidea sensu stricto is restricted to the families Galatheidae and Porcellanidae, and diagnoses for both Chirostyloidea and Galatheoidea are provided. Present results highlight the need for a detailed revision of a number of taxa, challenge some currently used morphological synapomorphies, and emphasise the need for integrated studies with wide taxon sampling and multiple data sources to resolve complex phylogenetic questions. Copyright © 2010 Elsevier Inc. All rights reserved.

S. pombe kinesins-8 promote both nucleation and catastrophe of microtubules.

Directory of Open Access Journals (Sweden)

Muriel Erent

Full Text Available The kinesins-8 were originally thought to be microtubule depolymerases, but are now emerging as more versatile catalysts of microtubule dynamics. We show here that S. pombe Klp5-436 and Klp6-440 are non-processive plus-end-directed motors whose in vitro velocities on S. pombe microtubules at 7 and 23 nm s(-1 are too slow to keep pace with the growing tips of dynamic interphase microtubules in living S. pombe. In vitro, Klp5 and 6 dimers exhibit a hitherto-undescribed combination of strong enhancement of microtubule nucleation with no effect on growth rate or catastrophe frequency. By contrast in vivo, both Klp5 and Klp6 promote microtubule catastrophe at cell ends whilst Klp6 also increases the number of interphase microtubule arrays (IMAs. Our data support a model in which Klp5/6 bind tightly to free tubulin heterodimers, strongly promoting the nucleation of new microtubules, and then continue to land as a tubulin-motor complex on the tips of growing microtubules, with the motors then dissociating after a few seconds residence on the lattice. In vivo, we predict that only at cell ends, when growing microtubule tips become lodged and their growth slows down, will Klp5/6 motor activity succeed in tracking growing microtubule tips. This mechanism would allow Klp5/6 to detect the arrival of microtubule tips at cells ends and to amplify the intrinsic tendency for microtubules to catastrophise in compression at cell ends. Our evidence identifies Klp5 and 6 as spatial regulators of microtubule dynamics that enhance both microtubule nucleation at the cell centre and microtubule catastrophe at the cell ends.

ATPase and GTPase Tangos Drive Intracellular Protein Transport.

Science.gov (United States)

Shan, Shu-Ou

2016-12-01

The GTPase superfamily of proteins provides molecular switches to regulate numerous cellular processes. The 'GTPase switch' paradigm, in which external regulatory factors control the switch of a GTPase between 'on' and 'off' states, has been used to interpret the regulatory mechanism of many GTPases. However, recent work unveiled a class of nucleotide hydrolases that do not adhere to this classical paradigm. Instead, they use nucleotide-dependent dimerization cycles to regulate key cellular processes. In this review article, recent studies of dimeric GTPases and ATPases involved in intracellular protein targeting are summarized. It is suggested that these proteins can use the conformational plasticity at their dimer interface to generate multiple points of regulation, thereby providing the driving force and spatiotemporal coordination of complex cellular pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism

DEFF Research Database (Denmark)

Sweeney, Trevor; Cisnetto, Valentina; Bose, Daniel

2010-01-01

Foot-and-mouth disease virus (FMDV), a positive sense, single-stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases a group of AAA+ ATPases that has a predicted N-termin...

RASOnD - A comprehensive resource and search tool for RAS superfamily oncogenes from various species

Directory of Open Access Journals (Sweden)

Singh Tej P

2011-07-01

Full Text Available Abstract Background The Ras superfamily plays an important role in the control of cell signalling and division. Mutations in the Ras genes convert them into active oncogenes. The Ras oncogenes form a major thrust of global cancer research as they are involved in the development and progression of tumors. This has resulted in the exponential growth of data on Ras superfamily across different public databases and in literature. However, no dedicated public resource is currently available for data mining and analysis on this family. The present database was developed to facilitate straightforward accession, retrieval and analysis of information available on Ras oncogenes from one particular site. Description We have developed the RAS Oncogene Database (RASOnD as a comprehensive knowledgebase that provides integrated and curated information on a single platform for oncogenes of Ras superfamily. RASOnD encompasses exhaustive genomics and proteomics data existing across diverse publicly accessible databases. This resource presently includes overall 199,046 entries from 101 different species. It provides a search tool to generate information about their nucleotide and amino acid sequences, single nucleotide polymorphisms, chromosome positions, orthologies, motifs, structures, related pathways and associated diseases. We have implemented a number of user-friendly search interfaces and sequence analysis tools. At present the user can (i browse the data (ii search any field through a simple or advance search interface and (iii perform a BLAST search and subsequently CLUSTALW multiple sequence alignment by selecting sequences of Ras oncogenes. The Generic gene browser, GBrowse, JMOL for structural visualization and TREEVIEW for phylograms have been integrated for clear perception of retrieved data. External links to related databases have been included in RASOnD. Conclusions This database is a resource and search tool dedicated to Ras oncogenes. It has

Labeling proteins inside living cells using external fluorophores for microscopy.

Science.gov (United States)

Teng, Kai Wen; Ishitsuka, Yuji; Ren, Pin; Youn, Yeoan; Deng, Xiang; Ge, Pinghua; Lee, Sang Hak; Belmont, Andrew S; Selvin, Paul R

2016-12-09

Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.

The triple helix of collagens - an ancient protein structure that enabled animal multicellularity and tissue evolution.

Science.gov (United States)

Fidler, Aaron L; Boudko, Sergei P; Rokas, Antonis; Hudson, Billy G

2018-04-09

The cellular microenvironment, characterized by an extracellular matrix (ECM), played an essential role in the transition from unicellularity to multicellularity in animals (metazoans), and in the subsequent evolution of diverse animal tissues and organs. A major ECM component are members of the collagen superfamily -comprising 28 types in vertebrates - that exist in diverse supramolecular assemblies ranging from networks to fibrils. Each assembly is characterized by a hallmark feature, a protein structure called a triple helix. A current gap in knowledge is understanding the mechanisms of how the triple helix encodes and utilizes information in building scaffolds on the outside of cells. Type IV collagen, recently revealed as the evolutionarily most ancient member of the collagen superfamily, serves as an archetype for a fresh view of fundamental structural features of a triple helix that underlie the diversity of biological activities of collagens. In this Opinion, we argue that the triple helix is a protein structure of fundamental importance in building the extracellular matrix, which enabled animal multicellularity and tissue evolution. © 2018. Published by The Company of Biologists Ltd.

Structural Elements Regulating AAA+ Protein Quality Control Machines.

Science.gov (United States)

Chang, Chiung-Wen; Lee, Sukyeong; Tsai, Francis T F

2017-01-01

Members of the ATPases Associated with various cellular Activities (AAA+) superfamily participate in essential and diverse cellular pathways in all kingdoms of life by harnessing the energy of ATP binding and hydrolysis to drive their biological functions. Although most AAA+ proteins share a ring-shaped architecture, AAA+ proteins have evolved distinct structural elements that are fine-tuned to their specific functions. A central question in the field is how ATP binding and hydrolysis are coupled to substrate translocation through the central channel of ring-forming AAA+ proteins. In this mini-review, we will discuss structural elements present in AAA+ proteins involved in protein quality control, drawing similarities to their known role in substrate interaction by AAA+ proteins involved in DNA translocation. Elements to be discussed include the pore loop-1, the Inter-Subunit Signaling (ISS) motif, and the Pre-Sensor I insert (PS-I) motif. Lastly, we will summarize our current understanding on the inter-relationship of those structural elements and propose a model how ATP binding and hydrolysis might be coupled to polypeptide translocation in protein quality control machines.

Fetal antigen 1 (FA1), a circulating member of the epidermal growth factor (EGF) superfamily

DEFF Research Database (Denmark)

Jensen, Charlotte Harken; Krogh, T N; Støving, René Klinkby

1997-01-01

We describe an ELISA technique for quantification of fetal antigen 1 (FA1), a glycoprotein belonging to the EGF-superfamily. The ELISA is based on immunospecifically purified polyclonal antibodies and has a dynamic range of 0.7-5.3 ng/ml, intra- and inter-assay C.V.s of less than 3.2% and an aver......We describe an ELISA technique for quantification of fetal antigen 1 (FA1), a glycoprotein belonging to the EGF-superfamily. The ELISA is based on immunospecifically purified polyclonal antibodies and has a dynamic range of 0.7-5.3 ng/ml, intra- and inter-assay C.V.s of less than 3.......2% and an average recovery of 105% in serum and 98% in urine. Comparison of FA1 in amniotic fluid, serum and urine revealed parallel titration curves, identical elution volumes following size chromatography, immunological identity and similar profiles when analysed by MALDI-MS. The reference interval for serum FA1...... was 12.3-46.6 ng/ml and the levels were 10 times higher in patients with renal failure. FA1 showed no diurnal variation, no variation during the menstrual cycle and was not influenced by the acute phase reaction. In humans (n = 10) the renal clearance of FA1 was 11 ml/min and an identical high renal...

Plant Glycine-Rich Proteins in Stress Response: An Emerging, Still Prospective Story

Directory of Open Access Journals (Sweden)

Magdalena Czolpinska

2018-03-01

Full Text Available Seed plants are sessile organisms that have developed a plethora of strategies for sensing, avoiding, and responding to stress. Several proteins, including the glycine-rich protein (GRP superfamily, are involved in cellular stress responses and signaling. GRPs are characterized by high glycine content and the presence of conserved segments including glycine-containing structural motifs composed of repetitive amino acid residues. The general structure of this superfamily facilitates division of GRPs into five main subclasses. Although the participation of GRPs in plant stress response has been indicated in numerous model and non-model plant species, relatively little is known about the key physiological processes and molecular mechanisms in which those proteins are engaged. Class I, II, and IV members are known to be involved in hormone signaling, stress acclimation, and floral development, and are crucial for regulation of plant cells growth. GRPs of class IV [RNA-binding proteins (RBPs] are involved in alternative splicing or regulation of transcription and stomatal movement, seed, pollen, and stamen development; their accumulation is regulated by the circadian clock. Owing to the fact that the overexpression of GRPs can confer tolerance to stress (e.g., some are involved in cold acclimation and may improve growth at low temperatures, these proteins could play a promising role in agriculture through plant genetic engineering. Consequently, isolation, cloning, characterization, and functional validation of novel GRPs expressed in response to the diverse stress conditions are expected to be growing areas of research in the coming years. According to our knowledge, this is the first comprehensive review on participation of plant GRPs in the response to diverse stress stimuli.

Classification of Rhizomonas suberifaciens, an unnamed Rhizomonas species, and Sphingomonas spp. in rRNA superfamily IV.

Science.gov (United States)

van Bruggen, A H; Jochimsen, K N; Steinberger, E M; Segers, P; Gillis, M

1993-01-01

Thermal melting profiles of hybrids between 3H-labeled rRNA of Rhizomonas suberifaciens, the causal agent of corky root of lettuce, and chromosomal DNAs from 27 species of gram-negative bacteria indicated that the genus Rhizomonas belongs to superfamily IV of De Ley. On the basis of the melting temperatures of DNA hybrids with rRNAs from the type strains of R. suberifaciens, Sphingomonas paucimobilis, and Sphingomonas capsulata, Rhizomonas strains constitute a separate branch in superfamily IV, which is closely related to but separate from branches containing Zymomonas mobilis, Sphingomonas spp., and S. capsulata. Sphingomonas yanoikuyae and Rhizomonas sp. strain WI4 are located toward the base of the Rhizomonas rRNA branch. DNA-DNA hybridization indicated that S. yanoikuyae is equidistant from Rhizomonas sp. strain WI4 and S. paucimobilis. Sequences of 270 bp of 16S ribosomal DNAs from eight strains of Rhizomonas spp., eight strains of Sphingomonas spp., and Agrobacterium tumefaciens indicated that S. yanoikuyae and Rhizomonas sp. strains WI4 and CA16 are genetically more closely related to R. suberifaciens than to Sphingomonas spp. Thus, S. yanoikuyae may need to be transferred to the genus Rhizomonas on the basis of the results of further study.

Photoclickable dendritic molecular glue: noncovalent-to-covalent photochemical transformation of protein hybrids.

Science.gov (United States)

Uchida, Noriyuki; Okuro, Kou; Niitani, Yamato; Ling, Xiao; Ariga, Takayuki; Tomishige, Michio; Aida, Takuzo

2013-03-27

A water-soluble dendron with a fluorescein isothiocyanate (FITC) fluorescent label and bearing nine pendant guanidinium ion (Gu(+))/benzophenone (BP) pairs at its periphery (Glue(BP)-FITC) serves as a "photoclickable molecular glue". By multivalent salt-bridge formation between Gu(+) ions and oxyanions, Glue(BP)-FITC temporarily adheres to a kinesin/microtubule hybrid. Upon subsequent exposure to UV light, this noncovalent binding is made permanent via a cross-linking reaction mediated by carbon radicals derived from the photoexcited BP units. This temporal-to-permanent transformation by light occurs quickly and efficiently in this preorganized state, allowing the movements of microtubules on a kinesin-coated glass plate to be photochemically controlled. A fundamental difference between such temporal and permanent bindings was visualized by the use of "optical tweezers".

Five Drosophila Genomes Reveal Nonneutral Evolution and the Signature of Host Specialization in the Chemoreceptor Superfamily

OpenAIRE

McBride, Carolyn S.; Arguello, J. Roman

2007-01-01

The insect chemoreceptor superfamily comprises the olfactory receptor (Or) and gustatory receptor (Gr) multigene families. These families give insects the ability to smell and taste chemicals in the environment and are thus rich resources for linking molecular evolutionary and ecological processes. Although dramatic differences in family size among distant species and high divergence among paralogs have led to the belief that the two families evolve rapidly, a lack of evolutionary data over s...

Reliability of nine programs of topological predictions and their application to integral membrane channel and carrier proteins.

Science.gov (United States)

Reddy, Abhinay; Cho, Jaehoon; Ling, Sam; Reddy, Vamsee; Shlykov, Maksim; Saier, Milton H

2014-01-01

We evaluated topological predictions for nine different programs, HMMTOP, TMHMM, SVMTOP, DAS, SOSUI, TOPCONS, PHOBIUS, MEMSAT-SVM (hereinafter referred to as MEMSAT), and SPOCTOPUS. These programs were first evaluated using four large topologically well-defined families of secondary transporters, and the three best programs were further evaluated using topologically more diverse families of channels and carriers. In the initial studies, the order of accuracy was: SPOCTOPUS > MEMSAT > HMMTOP > TOPCONS > PHOBIUS > TMHMM > SVMTOP > DAS > SOSUI. Some families, such as the Sugar Porter Family (2.A.1.1) of the Major Facilitator Superfamily (MFS; TC #2.A.1) and the Amino Acid/Polyamine/Organocation (APC) Family (TC #2.A.3), were correctly predicted with high accuracy while others, such as the Mitochondrial Carrier (MC) (TC #2.A.29) and the K(+) transporter (Trk) families (TC #2.A.38), were predicted with much lower accuracy. For small, topologically homogeneous families, SPOCTOPUS and MEMSAT were generally most reliable, while with large, more diverse superfamilies, HMMTOP often proved to have the greatest prediction accuracy. We next developed a novel program, TM-STATS, that tabulates HMMTOP, SPOCTOPUS or MEMSAT-based topological predictions for any subdivision (class, subclass, superfamily, family, subfamily, or any combination of these) of the Transporter Classification Database (TCDB; www.tcdb.org) and examined the following subclasses: α-type channel proteins (TC subclasses 1.A and 1.E), secreted pore-forming toxins (TC subclass 1.C) and secondary carriers (subclass 2.A). Histograms were generated for each of these subclasses, and the results were analyzed according to subclass, family and protein. The results provide an update of topological predictions for integral membrane transport proteins as well as guides for the development of more reliable topological prediction programs, taking family-specific characteristics into account. © 2014 S. Karger AG, Basel.

KBP interacts with SCG10, linking Goldberg-Shprintzen syndrome to microtubule dynamics and neuronal differentiation

NARCIS (Netherlands)

M.M. Alves (Maria); G.M. Burzynski (Grzegorz); J.-M. Delalande (Jean-Marie); J. Osinga (Jan); A. van der Goot (Annemieke); A.M. Dolga (Amalia); E. de Graaff (Esther); A.S. Brooks (Alice); M. Metzger (Marco); U.L.M. Eisel (Ulrich); I.T. Shepherd (Iain); B.J. Eggen (Bart); R.M.W. Hofstra (Robert)

2010-01-01

textabstractGoldberg-Shprintzen syndrome (GOSHS) is a rare clinical disorder characterized by central and enteric nervous system defects. This syndrome is caused by inactivating mutations in the Kinesin Binding Protein (KBP) gene, which encodes a protein of which the precise function is largely

Thermophilic P-loop transport ATPases : Enzyme function and energetics at high temperature

NARCIS (Netherlands)

Pretz, Monika Gyöngyi

2007-01-01

Primary transport ATPases are divided into several superfamilies; amongst others including ATPases of the ABC transporter superfamily, the F-ATPase superfamily or the motor ATPases of the General Secretory (Sec) pathway. Motor proteins from these superfamilies show a low sequence similarity, except

A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells

Directory of Open Access Journals (Sweden)

Amber L. Jolly

2016-01-01

Full Text Available Long-distance intracellular transport of organelles, mRNA, and proteins (“cargo” occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins, but the vast network of factors involved in regulating intracellular cargo transport are still unknown. We capitalize on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. We use an automated tracking program and a naive Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. We identify innate immunity genes, ion channels, and signaling proteins having a role in lysosome motility regulation and find an unexpected relationship between the dynein motor, Rab7a, and lysosome motility regulation.

Phylogenetic continuum indicates "galaxies" in the protein universe: preliminary results on the natural group structures of proteins.

Science.gov (United States)

Ladunga, I

1992-04-01

The markedly nonuniform, even systematic distribution of sequences in the protein "universe" has been analyzed by methods of protein taxonomy. Mapping of the natural hierarchical system of proteins has revealed some dense cores, i.e., well-defined clusterings of proteins that seem to be natural structural groupings, possibly seeds for a future protein taxonomy. The aim was not to force proteins into more or less man-made categories by discriminant analysis, but to find structurally similar groups, possibly of common evolutionary origin. Single-valued distance measures between pairs of superfamilies from the Protein Identification Resource were defined by two chi 2-like methods on tripeptide frequencies and the variable-length subsequence identity method derived from dot-matrix comparisons. Distance matrices were processed by several methods of cluster analysis to detect phylogenetic continuum between highly divergent proteins. Only well-defined clusters characterized by relatively unique structural, intracellular environmental, organismal, and functional attribute states were selected as major protein groups, including subsets of viral and Escherichia coli proteins, hormones, inhibitors, plant, ribosomal, serum and structural proteins, amino acid synthases, and clusters dominated by certain oxidoreductases and apolar and DNA-associated enzymes. The limited repertoire of functional patterns due to small genome size, the high rate of recombination, specific features of the bacterial membranes, or of the virus cycle canalize certain proteins of viruses and Gram-negative bacteria, respectively, to organismal groups.

ALDH16A1 is a novel non-catalytic enzyme that may be involved in the etiology of gout via protein-protein interactions with HPRT1.

Science.gov (United States)

Vasiliou, Vasilis; Sandoval, Monica; Backos, Donald S; Jackson, Brian C; Chen, Ying; Reigan, Philip; Lanaspa, Miguel A; Johnson, Richard J; Koppaka, Vindhya; Thompson, David C

2013-02-25

Gout, a common form of inflammatory arthritis, is strongly associated with elevated uric acid concentrations in the blood (hyperuricemia). A recent study in Icelanders identified a rare missense single nucleotide polymorphism (SNP) in the ALDH16A1 gene, ALDH16A1*2, to be associated with gout and serum uric acid levels. ALDH16A1 is a novel and rather unique member of the ALDH superfamily in relation to its gene and protein structures. ALDH16 genes are present in fish, amphibians, protista, bacteria but absent from archaea, fungi and plants. In most mammalian species, two ALDH16A1 spliced variants (ALDH16A1, long form and ALDH16A1_v2, short form) have been identified and both are expressed in HepG-2, HK-2 and HK-293 human cell lines. The ALDH16 proteins contain two ALDH domains (as opposed to one in the other members of the superfamily), four transmembrane and one coiled-coil domains. The active site of ALDH16 proteins from bacterial, frog and lower animals contain the catalytically important cysteine residue (Cys-302); this residue is absent from the mammalian and fish orthologs. Molecular modeling predicts that both the short and long forms of human ALDH16A1 protein would lack catalytic activity but may interact with the hypoxanthine-guanine phosphoribosyltransferase (HPRT1) protein, a key enzyme involved in uric acid metabolism and gout. Interestingly, such protein-protein interactions with HPRT1 are predicted to be impaired for the long or short forms of ALDH16A1*2. These results lead to the intriguing possibility that association between ALDH16A1 and HPRT1 may be required for optimal HPRT activity with disruption of this interaction possibly contributing to the hyperuricemia seen in ALDH16A1*2 carriers. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Mechanisms of EHD/RME-1 Protein Function in Endocytic Transport

Science.gov (United States)

Grant, Barth D.; Caplan, Steve

2009-01-01

The evolutionarily conserved Eps15 homology domain (EHD)/receptor-mediated endocytosis (RME)-1 family of C-terminal EH domain proteins has recently come under intense scrutiny because of its importance in intracellular membrane transport, especially with regard to the recycling of receptors from endosomes to the plasma membrane. Recent studies have shed new light on the mode by which these adenosine triphosphatases function on endosomal membranes in mammals and Caenorhabditis elegans. This review highlights our current understanding of the physiological roles of these proteins in vivo, discussing conserved features as well as emerging functional differences between individual mammalian paralogs. In addition, these findings are discussed in light of the identification of novel EHD/RME-1 protein and lipid interactions and new structural data for proteins in this family, indicating intriguing similarities to the Dynamin superfamily of large guanosine triphosphatases. PMID:18801062

New Insights into the Phylogeny and Gene Context Analysis of Binder of Sperm Proteins (BSPs.

Directory of Open Access Journals (Sweden)

Edith Serrano

Full Text Available Seminal plasma (SP proteins support the survival of spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, resulting in higher fertilizing ability. Among SP proteins, BSP (binder of sperm proteins are the most studied, since they may be useful for the improvement of semen diluents, storage and subsequent fertilization results. However, an updated and detailed phylogenetic analysis of the BSP protein superfamily has not been carried out with all the sequences described in the main databases. The update view shows for the first time an equally distributed number of sequences between the three families: BSP, and their homologs 1 (BSPH1 and 2 (BSPH2. The BSP family is divided in four subfamilies, BSP1 subfamily being the predominant, followed by subfamilies BSP3, BSP5 and BSP2. BSPH proteins were found among placental mammals (Eutheria belonging to the orders Proboscidea, Primates, Lagomorpha, Rodentia, Chiroptera, Perissodactyla and Cetartiodactyla. However, BSPH2 proteins were also found in the Scandentia order and Metatheria clade. This phylogenetic analysis, when combined with a gene context analysis, showed a completely new evolutionary scenario for the BSP superfamily of proteins with three defined different gene patterns, one for BSPs, one for BSPH1/BSPH2/ELSPBP1 and another one for BSPH1/BSPH2 without ELSPBP1. In addition, the study has permitted to define concise conserved blocks for each family (BSP, BSPH1 and BSPH2, which could be used for a more reliable assignment for the incoming sequences, for data curation of current databases, and for cloning new BSPs, as the one described in this paper, ram seminal vesicle 20 kDa protein (RSVP20, Ovis aries BSP5b.

The Ly6 protein coiled is required for septate junction and blood brain barrier organisation in Drosophila.

Science.gov (United States)

Hijazi, Assia; Haenlin, Marc; Waltzer, Lucas; Roch, Fernando

2011-03-15

Genetic analysis of the Drosophila septate junctions has greatly contributed to our understanding of the mechanisms controlling the assembly of these adhesion structures, which bear strong similarities with the vertebrate tight junctions and the paranodal septate junctions. These adhesion complexes share conserved molecular components and have a common function: the formation of paracellular barriers restraining the diffusion of solutes through epithelial and glial envelopes. In this work we characterise the function of the Drosophila cold gene, that codes for a protein belonging to the Ly6 superfamily of extracellular ligands. Analysis of cold mutants shows that this gene is specifically required for the organisation of the septate junctions in epithelial tissues and in the nervous system, where its contribution is essential for the maintenance of the blood-brain barrier. We show that cold acts in a cell autonomous way, and we present evidence indicating that this protein could act as a septate junction component. We discuss the specific roles of cold and three other Drosophila members of the Ly6 superfamily that have been shown to participate in a non-redundant way in the process of septate junction assembly. We propose that vertebrate Ly6 proteins could fulfill analogous roles in tight junctions and/or paranodal septate junctions.

KBP interacts with SCG10, linking Goldberg-Shprintzen syndrome to microtubule dynamics and neuronal differentiation

NARCIS (Netherlands)

Alves, Maria M.; Burzynski, Grzegorz; Delalande, Jean-Marie; Osinga, Jan; van der Goot, Annemieke; Dolga, Amalia; de Graaff, Esther; Brooks, Alice S.; Metzger, Marco; Eisel, Ulrich L.M.; Shepherd, Iain; Eggen, Bart J.L.; Hofstra, Robert M.W.

2010-01-01

Goldberg-Shprintzen syndrome (GOSHS) is a rare clinical disorder characterized by central and enteric nervous system defects. This syndrome is caused by inactivating mutations in the Kinesin Binding Protein (KBP) gene, which encodes a protein of which the precise function is largely unclear. We show

Maltose-binding protein effectively stabilizes the partially closed conformation of the ATP-binding cassette transporter MalFGK2

KAUST Repository

Weng, Jingwei; Gu, Shuo; Gao, Xin; Huang, Xuhui; Wang, Wenning

2017-01-01

Maltose transporter MalFGK2 is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily. Upon the binding of its periplasmic binding protein, MalE, the ATPase activity of MalFGK2 can be greatly enhanced. Crystal structures of the MalFGK2-MalE-maltose complex in a so-called

Maltose-binding protein effectively stabilizes the partially closed conformation of the ATP-binding cassette transporter MalFGK2

KAUST Repository

Weng, Jingwei

2017-02-23

Maltose transporter MalFGK2 is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily. Upon the binding of its periplasmic binding protein, MalE, the ATPase activity of MalFGK2 can be greatly enhanced. Crystal structures of the MalFGK2-MalE-maltose complex in a so-called

An expressed sequence tag (EST) data mining strategy succeeding in the discovery of new G-protein coupled receptors.

Science.gov (United States)

Wittenberger, T; Schaller, H C; Hellebrand, S

2001-03-30

We have developed a comprehensive expressed sequence tag database search method and used it for the identification of new members of the G-protein coupled receptor superfamily. Our approach proved to be especially useful for the detection of expressed sequence tag sequences that do not encode conserved parts of a protein, making it an ideal tool for the identification of members of divergent protein families or of protein parts without conserved domain structures in the expressed sequence tag database. At least 14 of the expressed sequence tags found with this strategy are promising candidates for new putative G-protein coupled receptors. Here, we describe the sequence and expression analysis of five new members of this receptor superfamily, namely GPR84, GPR86, GPR87, GPR90 and GPR91. We also studied the genomic structure and chromosomal localization of the respective genes applying in silico methods. A cluster of six closely related G-protein coupled receptors was found on the human chromosome 3q24-3q25. It consists of four orphan receptors (GPR86, GPR87, GPR91, and H963), the purinergic receptor P2Y1, and the uridine 5'-diphosphoglucose receptor KIAA0001. It seems likely that these receptors evolved from a common ancestor and therefore might have related ligands. In conclusion, we describe a data mining procedure that proved to be useful for the identification and first characterization of new genes and is well applicable for other gene families. Copyright 2001 Academic Press.

Topological variation in the evolution of new reactions in functionally diverse enzyme superfamilies.

Science.gov (United States)

Meng, Elaine C; Babbitt, Patricia C

2011-06-01

In functionally diverse enzyme superfamilies (SFs), conserved structural and active site features reflect catalytic capabilities 'hard-wired' in each SF architecture. Overlaid on this foundation, evolutionary changes in active site machinery, structural topology and other aspects of structural organization and interactions support the emergence of new reactions, mechanisms, and substrate specificity. This review connects topological with functional variation in each of the haloalkanoic acid dehalogenase (HAD) and vicinal oxygen chelate fold (VOC) SFs and a set of redox-active thioredoxin (Trx)-fold SFs to illustrate a few of the varied themes nature has used to evolve new functions from a limited set of structural scaffolds. Copyright © 2011 Elsevier Ltd. All rights reserved.

How to find soluble proteins: a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli

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Barth Sandra

2005-04-01

Full Text Available Abstract Background In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5:443–448. We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. Results The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. Conclusion A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used

Topology of membrane proteins-predictions, limitations and variations.

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Tsirigos, Konstantinos D; Govindarajan, Sudha; Bassot, Claudio; Västermark, Åke; Lamb, John; Shu, Nanjiang; Elofsson, Arne

2017-10-26

Transmembrane proteins perform a variety of important biological functions necessary for the survival and growth of the cells. Membrane proteins are built up by transmembrane segments that span the lipid bilayer. The segments can either be in the form of hydrophobic alpha-helices or beta-sheets which create a barrel. A fundamental aspect of the structure of transmembrane proteins is the membrane topology, that is, the number of transmembrane segments, their position in the protein sequence and their orientation in the membrane. Along these lines, many predictive algorithms for the prediction of the topology of alpha-helical and beta-barrel transmembrane proteins exist. The newest algorithms obtain an accuracy close to 80% both for alpha-helical and beta-barrel transmembrane proteins. However, lately it has been shown that the simplified picture presented when describing a protein family by its topology is limited. To demonstrate this, we highlight examples where the topology is either not conserved in a protein superfamily or where the structure cannot be described solely by the topology of a protein. The prediction of these non-standard features from sequence alone was not successful until the recent revolutionary progress in 3D-structure prediction of proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

The human protein disulfide isomerase gene family

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Galligan James J

2012-07-01

Full Text Available Abstract Enzyme-mediated disulfide bond formation is a highly conserved process affecting over one-third of all eukaryotic proteins. The enzymes primarily responsible for facilitating thiol-disulfide exchange are members of an expanding family of proteins known as protein disulfide isomerases (PDIs. These proteins are part of a larger superfamily of proteins known as the thioredoxin protein family (TRX. As members of the PDI family of proteins, all proteins contain a TRX-like structural domain and are predominantly expressed in the endoplasmic reticulum. Subcellular localization and the presence of a TRX domain, however, comprise the short list of distinguishing features required for gene family classification. To date, the PDI gene family contains 21 members, varying in domain composition, molecular weight, tissue expression, and cellular processing. Given their vital role in protein-folding, loss of PDI activity has been associated with the pathogenesis of numerous disease states, most commonly related to the unfolded protein response (UPR. Over the past decade, UPR has become a very attractive therapeutic target for multiple pathologies including Alzheimer disease, Parkinson disease, alcoholic and non-alcoholic liver disease, and type-2 diabetes. Understanding the mechanisms of protein-folding, specifically thiol-disulfide exchange, may lead to development of a novel class of therapeutics that would help alleviate a wide range of diseases by targeting the UPR.

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

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

2005-11-21

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

Characterization of bud emergence 46 (BEM46) protein: Sequence, structural, phylogenetic and subcellular localization analyses

International Nuclear Information System (INIS)

Kumar, Abhishek; Kollath-Leiß, Krisztina; Kempken, Frank

2013-01-01

Highlights: •All eukaryotes have at least a single copy of a bem46 ortholog. •The catalytic triad of BEM46 is illustrated using sequence and structural analysis. •We identified indels in the conserved domain of BEM46 protein. •Localization studies of BEM46 protein were carried out using GFP-fusion tagging. -- Abstract: The bud emergence 46 (BEM46) protein from Neurospora crassa belongs to the α/β-hydrolase superfamily. Recently, we have reported that the BEM46 protein is localized in the perinuclear ER and also forms spots close by the plasma membrane. The protein appears to be required for cell type-specific polarity formation in N. crassa. Furthermore, initial studies suggested that the BEM46 amino acid sequence is conserved in eukaryotes and is considered to be one of the widespread conserved “known unknown” eukaryotic genes. This warrants for a comprehensive phylogenetic analysis of this superfamily to unravel origin and molecular evolution of these genes in different eukaryotes. Herein, we observe that all eukaryotes have at least a single copy of a bem46 ortholog. Upon scanning of these proteins in various genomes, we find that there are expansions leading into several paralogs in vertebrates. Usingcomparative genomic analyses, we identified insertion/deletions (indels) in the conserved domain of BEM46 protein, which allow to differentiate fungal classes such as ascomycetes from basidiomycetes. We also find that exonic indels are able to differentiate BEM46 homologs of different eukaryotic lineage. Furthermore, we unravel that BEM46 protein from N. crassa possess a novel endoplasmic-retention signal (PEKK) using GFP-fusion tagging experiments. We propose that three residues namely a serine 188S, a histidine 292H and an aspartic acid 262D are most critical residues, forming a catalytic triad in BEM46 protein from N. crassa. We carried out a comprehensive study on bem46 genes from a molecular evolution perspective with combination of functional

Characterization of bud emergence 46 (BEM46) protein: Sequence, structural, phylogenetic and subcellular localization analyses

Energy Technology Data Exchange (ETDEWEB)

Kumar, Abhishek; Kollath-Leiß, Krisztina; Kempken, Frank, E-mail: fkempken@bot.uni-kiel.de

2013-08-30

Highlights: •All eukaryotes have at least a single copy of a bem46 ortholog. •The catalytic triad of BEM46 is illustrated using sequence and structural analysis. •We identified indels in the conserved domain of BEM46 protein. •Localization studies of BEM46 protein were carried out using GFP-fusion tagging. -- Abstract: The bud emergence 46 (BEM46) protein from Neurospora crassa belongs to the α/β-hydrolase superfamily. Recently, we have reported that the BEM46 protein is localized in the perinuclear ER and also forms spots close by the plasma membrane. The protein appears to be required for cell type-specific polarity formation in N. crassa. Furthermore, initial studies suggested that the BEM46 amino acid sequence is conserved in eukaryotes and is considered to be one of the widespread conserved “known unknown” eukaryotic genes. This warrants for a comprehensive phylogenetic analysis of this superfamily to unravel origin and molecular evolution of these genes in different eukaryotes. Herein, we observe that all eukaryotes have at least a single copy of a bem46 ortholog. Upon scanning of these proteins in various genomes, we find that there are expansions leading into several paralogs in vertebrates. Usingcomparative genomic analyses, we identified insertion/deletions (indels) in the conserved domain of BEM46 protein, which allow to differentiate fungal classes such as ascomycetes from basidiomycetes. We also find that exonic indels are able to differentiate BEM46 homologs of different eukaryotic lineage. Furthermore, we unravel that BEM46 protein from N. crassa possess a novel endoplasmic-retention signal (PEKK) using GFP-fusion tagging experiments. We propose that three residues namely a serine 188S, a histidine 292H and an aspartic acid 262D are most critical residues, forming a catalytic triad in BEM46 protein from N. crassa. We carried out a comprehensive study on bem46 genes from a molecular evolution perspective with combination of functional

Genome-wide identification and analysis of the aldehyde dehydrogenase (ALDH) gene superfamily in apple (Malus × domestica Borkh.).

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Li, Xiaoqin; Guo, Rongrong; Li, Jun; Singer, Stacy D; Zhang, Yucheng; Yin, Xiangjing; Zheng, Yi; Fan, Chonghui; Wang, Xiping

2013-10-01

Aldehyde dehydrogenases (ALDHs) represent a protein superfamily encoding NAD(P)(+)-dependent enzymes that oxidize a wide range of endogenous and exogenous aliphatic and aromatic aldehydes. In plants, they are involved in many biological processes and play a role in the response to environmental stress. In this study, a total of 39 ALDH genes from ten families were identified in the apple (Malus × domestica Borkh.) genome. Synteny analysis of the apple ALDH (MdALDH) genes indicated that segmental and tandem duplications, as well as whole genome duplications, have likely contributed to the expansion and evolution of these gene families in apple. Moreover, synteny analysis between apple and Arabidopsis demonstrated that several MdALDH genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes appeared before the divergence of lineages that led to apple and Arabidopsis. In addition, phylogenetic analysis, as well as comparisons of exon-intron and protein structures, provided further insight into both their evolutionary relationships and their putative functions. Tissue-specific expression analysis of the MdALDH genes demonstrated diverse spatiotemporal expression patterns, while their expression profiles under abiotic stress and various hormone treatments indicated that many MdALDH genes were responsive to high salinity and drought, as well as different plant hormones. This genome-wide identification, as well as characterization of evolutionary relationships and expression profiles, of the apple MdALDH genes will not only be useful for the further analysis of ALDH genes and their roles in stress response, but may also aid in the future improvement of apple stress tolerance. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Computation-Facilitated Assignment of Function in the Enolase Superfamily: A Regiochemically Distinct Galactarate Dehydratase from Oceanobacillus iheyensis†

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Rakus, John F.; Kalyanaraman, Chakrapani; Fedorov, Alexander A.; Fedorov, Elena V.; Mills-Groninger, Fiona P.; Toro, Rafael; Bonanno, Jeffrey; Bain, Kevin; Sauder, J. Michael; Burley, Stephen K.; Almo, Steven C.; Jacobson, Matthew P.; Gerlt, John A.

2009-01-01

The structure of an uncharacterized member of the enolase superfamily from Oceanobacillus iheyensis (GI: 23100298; IMG locus tag Ob2843; PDB Code 2OQY) was determined by the New York SGX Research Center for Structural Genomics (NYSGXRC). The structure contained two Mg2+ ions located 10.4 Å from one another, with one located in the canonical position in the (β/α)7β-barrel domain (although the ligand at the end of the fifth β-strand is His, unprecedented in structurally characterized members of the superfamily); the second is located in a novel site within the capping domain. In silico docking of a library of mono- and diacid sugars to the active site predicted a diacid sugar as a likely substrate. Activity screening of a physical library of acid sugars identified galactarate as the substrate (kcat = 6.8 s−1, KM = 620 μM; kcat/KM = 1.1 × 104 M−1 s−1), allowing functional assignment of Ob2843 as galactarate dehydratase (GalrD-II) The structure of a complex of the catalytically impaired Y90F mutant with Mg2+ and galactarate allowed identification of a Tyr 164-Arg 162 dyad as the base that initiates the reaction by abstraction of the α-proton and Tyr 90 as the acid that facilitates departure of the β-OH leaving group. The enzyme product is 2-keto-3-deoxy-D-threo-4,5-dihydroxyadipate, the enantiomer of the product obtained in the GalrD reaction catalyzed by a previously characterized bifunctional L-talarate/galactarate dehydratase (TalrD/GalrD). On the basis of the different active site structures and different regiochemistries, we recognize that these functions represent an example of apparent, not actual, convergent evolution of function. The structure of GalrD-II and its active site architecture allow identification of the seventh functionally and structurally characterized subgroup in the enolase superfamily. This study provides an additional example that an integrated sequence/structure-based strategy employing computational approaches is a viable

Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends

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Ti, Shih-Chieh; Pamula, Melissa C.; Howes, Stuart C.; Duellberg, Christian; Cade, Nicholas I.; Kleiner, Ralph E.; Forth, Scott; Surrey, Thomas; Nogales, Eva; Kapoor, Tarun M.

2016-04-01

The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. In this research, we have purified and characterized tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.

NCBI nr-aa BLAST: CBRC-PHAM-01-0469 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-PHAM-01-0469 ref|ZP_04755732.1| fucose permease-like protein [Francisella philomiragia subsp. philo...miragia ATCC 25015] ref|ZP_05249399.1| major facilitator superfamily transporter [Francisella philo...miragia subsp. philomiragia ATCC 25015] gb|ACA66108.1| major facilitator superfamily protein [Francisella philo...miragia subsp. philomiragia] gb|EET21124.1| major facilitator supe...rfamily transporter [Francisella philomiragia subsp. philomiragia ATCC 25015] ZP_04755732.1 0.099 25% ...

Cross-over between discrete and continuous protein structure space: insights into automatic classification and networks of protein structures.

Directory of Open Access Journals (Sweden)

Alberto Pascual-García

2009-03-01

. As a domain set, we have selected a consensus set of 2,890 domains decomposed very similarly in SCOP and CATH. As an alignment algorithm, we used a global version of MAMMOTH developed in our group, which is both rapid and accurate. As a similarity measure, we used the size-normalized contact overlap, and as a clustering algorithm, we used average linkage. The resulting automatic classification at the cross-over point was more consistent than expert ones with respect to the structure similarity measure, with 86% of the clusters corresponding to subsets of either SCOP or CATH superfamilies and fewer than 5% containing domains in distinct folds according to both SCOP and CATH. Almost 15% of SCOP superfamilies and 10% of CATH superfamilies were split, consistent with the notion of fold change in protein evolution. These results were qualitatively robust for all choices that we tested, although we did not try to use alignment algorithms developed by other groups. Folds defined in SCOP and CATH would be completely joined in the regime of large transitivity violations where clustering is more arbitrary. Consistently, the agreement between SCOP and CATH at fold level was lower than their agreement with the automatic classification obtained using as a clustering algorithm, respectively, average linkage (for SCOP or single linkage (for CATH. The networks representing significant evolutionary and structural relationships between clusters beyond the cross-over point may allow us to perform evolutionary, structural, or functional analyses beyond the limits of classification schemes. These networks and the underlying clusters are available at http://ub.cbm.uam.es/research/ProtNet.php.

Plus- and minus-end directed microtubule motors bind simultaneously to herpes simplex virus capsids using different inner tegument structures.

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Kerstin Radtke

2010-07-01

Full Text Available Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1 show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during

Coin Tossing Explains the Activity of Opposing Microtubule Motors on Phagosomes.

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Sanghavi, Paulomi; D'Souza, Ashwin; Rai, Ashim; Rai, Arpan; Padinhatheeri, Ranjith; Mallik, Roop

2018-05-07

How the opposing activity of kinesin and dynein motors generates polarized distribution of organelles inside cells is poorly understood and hotly debated [1, 2]. Possible explanations include stochastic mechanical competition [3, 4], coordinated regulation by motor-associated proteins [5-7], mechanical activation of motors [8], and lipid-induced organization [9]. Here, we address this question by using phagocytosed latex beads to generate early phagosomes (EPs) that move bidirectionally along microtubules (MTs) in an in vitro assay [9]. Dynein/kinesin activity on individual EPs is recorded as real-time force generation of the motors against an optical trap. Activity of one class of motors frequently coincides with, or is rapidly followed by opposite motors. This leads to frequent and rapid reversals of EPs in the trap. Remarkably, the choice between dynein and kinesin can be explained by the tossing of a coin. Opposing motors therefore appear to function stochastically and independently of each other, as also confirmed by observing no effect on kinesin function when dynein is inhibited on the EPs. A simple binomial probability calculation based on the geometry of EP-microtubule contact explains the observed activity of dynein and kinesin on phagosomes. This understanding of intracellular transport in terms of a hypothetical coin, if it holds true for other cargoes, provides a conceptual framework to explain the polarized localization of organelles inside cells. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Melanophore migration and survival during zebrafish adult pigment stripe development require the immunoglobulin superfamily adhesion molecule Igsf11.

Science.gov (United States)

Eom, Dae Seok; Inoue, Shinya; Patterson, Larissa B; Gordon, Tiffany N; Slingwine, Rebecca; Kondo, Shigeru; Watanabe, Masakatsu; Parichy, David M

2012-01-01

The zebrafish adult pigment pattern has emerged as a useful model for understanding the development and evolution of adult form as well as pattern-forming mechanisms more generally. In this species, a series of horizontal melanophore stripes arises during the larval-to-adult transformation, but the genetic and cellular bases for stripe formation remain largely unknown. Here, we show that the seurat mutant phenotype, consisting of an irregular spotted pattern, arises from lesions in the gene encoding Immunoglobulin superfamily member 11 (Igsf11). We find that Igsf11 is expressed by melanophores and their precursors, and we demonstrate by cell transplantation and genetic rescue that igsf11 functions autonomously to this lineage in promoting adult stripe development. Further analyses of cell behaviors in vitro, in vivo, and in explant cultures ex vivo demonstrate that Igsf11 mediates adhesive interactions and that mutants for igsf11 exhibit defects in both the migration and survival of melanophores and their precursors. These findings identify the first in vivo requirements for igsf11 as well as the first instance of an immunoglobulin superfamily member functioning in pigment cell development and patterning. Our results provide new insights into adult pigment pattern morphogenesis and how cellular interactions mediate pattern formation.

Melanophore migration and survival during zebrafish adult pigment stripe development require the immunoglobulin superfamily adhesion molecule Igsf11.

Directory of Open Access Journals (Sweden)

Dae Seok Eom

Full Text Available The zebrafish adult pigment pattern has emerged as a useful model for understanding the development and evolution of adult form as well as pattern-forming mechanisms more generally. In this species, a series of horizontal melanophore stripes arises during the larval-to-adult transformation, but the genetic and cellular bases for stripe formation remain largely unknown. Here, we show that the seurat mutant phenotype, consisting of an irregular spotted pattern, arises from lesions in the gene encoding Immunoglobulin superfamily member 11 (Igsf11. We find that Igsf11 is expressed by melanophores and their precursors, and we demonstrate by cell transplantation and genetic rescue that igsf11 functions autonomously to this lineage in promoting adult stripe development. Further analyses of cell behaviors in vitro, in vivo, and in explant cultures ex vivo demonstrate that Igsf11 mediates adhesive interactions and that mutants for igsf11 exhibit defects in both the migration and survival of melanophores and their precursors. These findings identify the first in vivo requirements for igsf11 as well as the first instance of an immunoglobulin superfamily member functioning in pigment cell development and patterning. Our results provide new insights into adult pigment pattern morphogenesis and how cellular interactions mediate pattern formation.

The Ly6 protein coiled is required for septate junction and blood brain barrier organisation in Drosophila.

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Assia Hijazi

Full Text Available BACKGROUND: Genetic analysis of the Drosophila septate junctions has greatly contributed to our understanding of the mechanisms controlling the assembly of these adhesion structures, which bear strong similarities with the vertebrate tight junctions and the paranodal septate junctions. These adhesion complexes share conserved molecular components and have a common function: the formation of paracellular barriers restraining the diffusion of solutes through epithelial and glial envelopes. METHODOLOGY/PRINCIPAL FINDINGS: In this work we characterise the function of the Drosophila cold gene, that codes for a protein belonging to the Ly6 superfamily of extracellular ligands. Analysis of cold mutants shows that this gene is specifically required for the organisation of the septate junctions in epithelial tissues and in the nervous system, where its contribution is essential for the maintenance of the blood-brain barrier. We show that cold acts in a cell autonomous way, and we present evidence indicating that this protein could act as a septate junction component. CONCLUSION/SIGNIFICANCE: We discuss the specific roles of cold and three other Drosophila members of the Ly6 superfamily that have been shown to participate in a non-redundant way in the process of septate junction assembly. We propose that vertebrate Ly6 proteins could fulfill analogous roles in tight junctions and/or paranodal septate junctions.

New method to analyze super-family events observed with emulsion chambers

International Nuclear Information System (INIS)

Amenomori, M.

1997-01-01

The authors have developed a clustering method to analyze family events observed with emulsion chambers at high mountains. The main purpose of this analysis is to estimate the main production height of individual events, angular spread of gamma-rays in each event and so on. These enable them to investigate hadronic interactions at energies over 10 16 eV inaccessible by the present high-energy accelerators. they examined their clustering method using Monte Carlo events, and found that for the family events whose production height is low (within 2-3 km above the observation point in air), their production heights and lateral spreads are well reproduced. They further applied their method to the super-family events (ΣE γ > 1000 TeV) observed with emulsion chambers at Mt. Kanbala (5500 m above sea-level). The results seem to suggest that particle production with large transverse momentum occurs with considerable frequency even in the fragmentation region in the energy region over 10 16 eV

The structure of BVU2987 from Bacteroides vulgatus reveals a superfamily of bacterial periplasmic proteins with possible inhibitory function

International Nuclear Information System (INIS)

Das, Debanu; Finn, Robert D.; Carlton, Dennis; Miller, Mitchell D.; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Bakolitsa, Constantina; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Ernst, Dustin; Farr, Carol L.; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; Bedem, Henry van den; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

2010-01-01

The crystal structure of the BVU2987 gene product from B. vulgatus (UniProt A6L4L1) reveals that members of the new Pfam family PF11396 (domain of unknown function; DUF2874) are similar to β-lactamase inhibitor protein and YpmB. Proteins that contain the DUF2874 domain constitute a new Pfam family PF11396. Members of this family have predominantly been identified in microbes found in the human gut and oral cavity. The crystal structure of one member of this family, BVU2987 from Bacteroides vulgatus, has been determined, revealing a β-lactamase inhibitor protein-like structure with a tandem repeat of domains. Sequence analysis and structural comparisons reveal that BVU2987 and other DUF2874 proteins are related to β-lactamase inhibitor protein, PepSY and SmpA-OmlA proteins and hence are likely to function as inhibitory proteins

Regulatory crosstalk by protein kinases on CFTR trafficking and activity

Science.gov (United States)

Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

2016-01-01

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

NCBI nr-aa BLAST: CBRC-MDOM-01-0302 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-MDOM-01-0302 ref|ZP_05500348.1| RarD protein, DMT superfamily transporter [Thiomonas intermedia... K12] gb|EEU54627.1| RarD protein, DMT superfamily transporter [Thiomonas intermedia K12] ZP_05500348.1 1.9 27% ...

Structure of a cupin protein Plu4264 from Photorhabdus luminescens subsp. laumondii TTO1 at 1.35 Å resolution: Cupin Structure from Photorhabdus luminescens

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Weerth, R. Sophia [Department of Bacteriology, University of Wisconsin-Madison, Madison Wisconsin; Michalska, Karolina [Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, Argonne Illinois; Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne Illinois; Bingman, Craig A. [Department of Biochemistry, University of Wisconsin-Madison, Madison Wisconsin; Yennamalli, Ragothaman M. [Biosciences at Rice, Rice University, Houston Texas; Li, Hui [Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne Illinois; Jedrzejczak, Robert [Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne Illinois; Wang, Fengbin [Biosciences at Rice, Rice University, Houston Texas; Babnigg, Gyorgy [Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, Argonne Illinois; Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne Illinois; Joachimiak, Andrzej [Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, Argonne Illinois; Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne Illinois; Thomas, Michael G. [Department of Bacteriology, University of Wisconsin-Madison, Madison Wisconsin; Phillips, George N. [Biosciences at Rice, Rice University, Houston Texas

2014-12-18

Proteins belonging to the cupin superfamily have a wide range of catalytic and noncatalytic functions. Cupin proteins commonly have the capacity to bind a metal ion with the metal frequently determining the function of the protein. We have been investigating the function of homologous cupin proteins that are conserved in more than 40 species of bacteria. To gain insights into the potential function of these proteins we have solved the structure of Plu4264 from Photorhabdus luminescens TTO1 at a resolution of 1.35 Å and identified manganese as the likely natural metal ligand of the protein.

Understanding and Manipulating Electrostatic Fields at the Protein-Protein Interface Using Vibrational Spectroscopy and Continuum Electrostatics Calculations.

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Ritchie, Andrew W; Webb, Lauren J

2015-11-05

Biological function emerges in large part from the interactions of biomacromolecules in the complex and dynamic environment of the living cell. For this reason, macromolecular interactions in biological systems are now a major focus of interest throughout the biochemical and biophysical communities. The affinity and specificity of macromolecular interactions are the result of both structural and electrostatic factors. Significant advances have been made in characterizing structural features of stable protein-protein interfaces through the techniques of modern structural biology, but much less is understood about how electrostatic factors promote and stabilize specific functional macromolecular interactions over all possible choices presented to a given molecule in a crowded environment. In this Feature Article, we describe how vibrational Stark effect (VSE) spectroscopy is being applied to measure electrostatic fields at protein-protein interfaces, focusing on measurements of guanosine triphosphate (GTP)-binding proteins of the Ras superfamily binding with structurally related but functionally distinct downstream effector proteins. In VSE spectroscopy, spectral shifts of a probe oscillator's energy are related directly to that probe's local electrostatic environment. By performing this experiment repeatedly throughout a protein-protein interface, an experimental map of measured electrostatic fields generated at that interface is determined. These data can be used to rationalize selective binding of similarly structured proteins in both in vitro and in vivo environments. Furthermore, these data can be used to compare to computational predictions of electrostatic fields to explore the level of simulation detail that is necessary to accurately predict our experimental findings.

AAA+ Machines of Protein Destruction in Mycobacteria.

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Alhuwaider, Adnan Ali H; Dougan, David A

2017-01-01

The bacterial cytosol is a complex mixture of macromolecules (proteins, DNA, and RNA), which collectively are responsible for an enormous array of cellular tasks. Proteins are central to most, if not all, of these tasks and as such their maintenance (commonly referred to as protein homeostasis or proteostasis) is vital for cell survival during normal and stressful conditions. The two key aspects of protein homeostasis are, (i) the correct folding and assembly of proteins (coupled with their delivery to the correct cellular location) and (ii) the timely removal of unwanted or damaged proteins from the cell, which are performed by molecular chaperones and proteases, respectively. A major class of proteins that contribute to both of these tasks are the AAA+ (ATPases associated with a variety of cellular activities) protein superfamily. Although much is known about the structure of these machines and how they function in the model Gram-negative bacterium Escherichia coli , we are only just beginning to discover the molecular details of these machines and how they function in mycobacteria. Here we review the different AAA+ machines, that contribute to proteostasis in mycobacteria. Primarily we will focus on the recent advances in the structure and function of AAA+ proteases, the substrates they recognize and the cellular pathways they control. Finally, we will discuss the recent developments related to these machines as novel drug targets.

MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies

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2014-01-01

Background The development of chemical refolding of transforming growth factor-beta (TGF-β) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-β superfamily ligands could not be refolded readily by the same methods. Results Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 – Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. Conclusion MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of

S-nitrosylated proteins of a medicinal CAM plant Kalanchoe pinnata- ribulose-1,5-bisphosphate carboxylase/oxygenase activity targeted for inhibition.

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Abat, Jasmeet K; Mattoo, Autar K; Deswal, Renu

2008-06-01

Nitric oxide (NO) is a signaling molecule that affects a myriad of processes in plants. However, the mechanistic details are limited. NO post-translationally modifies proteins by S-nitrosylation of cysteines. The soluble S-nitrosoproteome of a medicinal, crassulacean acid metabolism (CAM) plant, Kalanchoe pinnata, was purified using the biotin switch technique. Nineteen targets were identified by MALDI-TOF mass spectrometry, including proteins associated with carbon, nitrogen and sulfur metabolism, the cytoskeleton, stress and photosynthesis. Some were similar to those previously identified in Arabidopsis thaliana, but kinesin-like protein, glycolate oxidase, putative UDP glucose 4-epimerase and putative DNA topoisomerase II had not been identified as targets previously for any organism. In vitro and in vivo nitrosylation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), one of the targets, was confirmed by immunoblotting. Rubisco plays a central role in photosynthesis, and the effect of S-nitrosylation on its enzymatic activity was determined using NaH14CO3. The NO-releasing compound S-nitrosoglutathione inhibited its activity in a dose-dependent manner suggesting Rubisco inactivation by nitrosylation for the first time.

Identification of TOEFAZ1-interacting proteins reveals key regulators of Trypanosoma brucei cytokinesis.

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Hilton, Nicholas A; Sladewski, Thomas E; Perry, Jenna A; Pataki, Zemplen; Sinclair-Davis, Amy N; Muniz, Richard S; Tran, Holly L; Wurster, Jenna I; Seo, Jiwon; de Graffenried, Christopher L

2018-05-21

The protist parasite Trypanosoma brucei is an obligate extracellular pathogen that retains its highly-polarized morphology during cell division and has evolved a novel cytokinetic process independent of non-muscle myosin II. The polo-like kinase homolog TbPLK is essential for transmission of cell polarity during division and for cytokinesis. We previously identified a putative TbPLK substrate named Tip of the Extending FAZ 1 (TOEFAZ1) as an essential kinetoplastid-specific component of the T. brucei cytokinetic machinery. We performed a proximity-dependent biotinylation (BioID) screen using TOEFAZ1 as a means to identify additional proteins that are involved in cytokinesis. Using quantitative proteomic methods, we identified nearly 500 TOEFAZ1-proximal proteins and characterized 59 in further detail. Among the candidates, we identified an essential putative phosphatase that regulates the expression level and localization of both TOEFAZ1 and TbPLK, a previously uncharacterized protein that is necessary for the assembly of a new cell posterior, and a microtubule plus-end directed orphan kinesin that is required for completing cleavage furrow ingression. The identification of these proteins provides new insight into T. brucei cytokinesis and establishes TOEFAZ1 as a key component of this essential and uniquely-configured process in kinetoplastids. This article is protected by copyright. All rights reserved. © 2018 John Wiley & Sons Ltd.

Characterization of Danio rerio Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase, the structural prototype of the ADPRibase-Mn-like protein family.

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Joaquim Rui Rodrigues

Full Text Available The ADPRibase-Mn-like protein family, that belongs to the metallo-dependent phosphatase superfamily, has different functional and structural prototypes. The functional one is the Mn(2+-dependent ADP-ribose/CDP-alcohol diphosphatase from Rattus norvegicus, which is essentially inactive with Mg(2+ and active with low micromolar Mn(2+ in the hydrolysis of the phosphoanhydride linkages of ADP-ribose, CDP-alcohols and cyclic ADP-ribose (cADPR in order of decreasing efficiency. The structural prototype of the family is a Danio rerio protein with a known crystallographic structure but functionally uncharacterized. To estimate the structure-function correlation with the same protein, the activities of zebrafish ADPRibase-Mn were studied. Differences between zebrafish and rat enzymes are highlighted. The former showed a complex activity dependence on Mn(2+, significant (≈25% Mg(2+-dependent activity, but was almost inactive on cADPR (150-fold less efficient than the rat counterpart. The low cADPR hydrolase activity agreed with the zebrafish genome lacking genes coding for proteins with significant homology with cADPR-forming enzymes. Substrate-docking to zebrafish wild-type protein, and characterization of the ADPRibase-Mn H97A mutant pointed to a role of His-97 in catalysis by orientation, and to a bidentate water bridging the dinuclear metal center as the potential nucleophile. Finally, three structural elements that delimit the active site entrance in the zebrafish protein were identified as unique to the ADPRibase-Mn-like family within the metallo-dependent phosphatase superfamily.

NCBI nr-aa BLAST: CBRC-BTAU-01-0582 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-BTAU-01-0582 ref|ZP_01115488.1| Permease of the major facilitator superfamily protein [Rein...ekea sp. MED297] gb|EAR08550.1| Permease of the major facilitator superfamily protein [Reinekea sp. MED297] ZP_01115488.1 0.003 29% ...

CD177: A member of the Ly-6 gene superfamily involved with neutrophil proliferation and polycythemia vera

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Bettinotti Maria

2004-03-01

Full Text Available Abstract Genes in the Leukocyte Antigen 6 (Ly-6 superfamily encode glycosyl-phosphatidylinositol (GPI anchored glycoproteins (gp with conserved domains of 70 to 100 amino acids and 8 to 10 cysteine residues. Murine Ly-6 genes encode important lymphocyte and hematopoietic stem cell antigens. Recently, a new member of the human Ly-6 gene superfamily has been described, CD177. CD177 is polymorphic and has at least two alleles, PRV-1 and NB1. CD177 was first described as PRV-1, a gene that is overexpressed in neutrophils from approximately 95% of patients with polycythemia vera and from about half of patients with essential thrombocythemia. CD177 encodes NB1 gp, a 58–64 kD GPI gp that is expressed by neutrophils and neutrophil precursors. NB1 gp carries Human Neutrophil Antigen (HNA-2a. Investigators working to identify the gene encoding NB1 gp called the CD177 allele they described NB1. NB1 gp is unusual in that neutrophils from some healthy people lack the NB1 gp completely and in most people NB1 gp is expressed by a subpopulation of neutrophils. The function of NB1 gp and the role of CD177 in the pathogenesis and clinical course of polycythemia vera and essential thrombocythemia are not yet known. However, measuring neutrophil CD177 mRNA levels has become an important marker for diagnosing the myeloproliferative disorders polycythemia vera and essential thrombocythemia.

Identification of similar regions of protein structures using integrated sequence and structure analysis tools

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Heiland Randy

2006-03-01

Full Text Available Abstract Background Understanding protein function from its structure is a challenging problem. Sequence based approaches for finding homology have broad use for annotation of both structure and function. 3D structural information of protein domains and their interactions provide a complementary view to structure function relationships to sequence information. We have developed a web site http://www.sblest.org/ and an API of web services that enables users to submit protein structures and identify statistically significant neighbors and the underlying structural environments that make that match using a suite of sequence and structure analysis tools. To do this, we have integrated S-BLEST, PSI-BLAST and HMMer based superfamily predictions to give a unique integrated view to prediction of SCOP superfamilies, EC number, and GO term, as well as identification of the protein structural environments that are associated with that prediction. Additionally, we have extended UCSF Chimera and PyMOL to support our web services, so that users can characterize their own proteins of interest. Results Users are able to submit their own queries or use a structure already in the PDB. Currently the databases that a user can query include the popular structural datasets ASTRAL 40 v1.69, ASTRAL 95 v1.69, CLUSTER50, CLUSTER70 and CLUSTER90 and PDBSELECT25. The results can be downloaded directly from the site and include function prediction, analysis of the most conserved environments and automated annotation of query proteins. These results reflect both the hits found with PSI-BLAST, HMMer and with S-BLEST. We have evaluated how well annotation transfer can be performed on SCOP ID's, Gene Ontology (GO ID's and EC Numbers. The method is very efficient and totally automated, generally taking around fifteen minutes for a 400 residue protein. Conclusion With structural genomics initiatives determining structures with little, if any, functional characterization

Structural and functional analysis of VQ motif-containing proteins in Arabidopsis as interacting proteins of WRKY transcription factors.

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Cheng, Yuan; Zhou, Yuan; Yang, Yan; Chi, Ying-Jun; Zhou, Jie; Chen, Jian-Ye; Wang, Fei; Fan, Baofang; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan; Chen, Zhixiang

2012-06-01

WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Recently, several groups have reported that proteins containing a short VQ (FxxxVQxLTG) motif interact with WRKY proteins. We have recently discovered that two VQ proteins from Arabidopsis (Arabidopsis thaliana), SIGMA FACTOR-INTERACTING PROTEIN1 and SIGMA FACTOR-INTERACTING PROTEIN2, act as coactivators of WRKY33 in plant defense by specifically recognizing the C-terminal WRKY domain and stimulating the DNA-binding activity of WRKY33. In this study, we have analyzed the entire family of 34 structurally divergent VQ proteins from Arabidopsis. Yeast (Saccharomyces cerevisiae) two-hybrid assays showed that Arabidopsis VQ proteins interacted specifically with the C-terminal WRKY domains of group I and the sole WRKY domains of group IIc WRKY proteins. Using site-directed mutagenesis, we identified structural features of these two closely related groups of WRKY domains that are critical for interaction with VQ proteins. Quantitative reverse transcription polymerase chain reaction revealed that expression of a majority of Arabidopsis VQ genes was responsive to pathogen infection and salicylic acid treatment. Functional analysis using both knockout mutants and overexpression lines revealed strong phenotypes in growth, development, and susceptibility to pathogen infection. Altered phenotypes were substantially enhanced through cooverexpression of genes encoding interacting VQ and WRKY proteins. These findings indicate that VQ proteins play an important role in plant growth, development, and response to environmental conditions, most likely by acting as cofactors of group I and IIc WRKY transcription factors.

Motor protein traffic regulation by supply–demand balance of resources

International Nuclear Information System (INIS)

Ciandrini, Luca; Dauloudet, Olivier; Parmeggiani, Andrea; Neri, Izaak; Walter, Jean Charles

2014-01-01

In cells and in in vitro assays the number of motor proteins involved in biological transport processes is far from being unlimited. The cytoskeletal binding sites are in contact with the same finite reservoir of motors (either the cytosol or the flow chamber) and hence compete for recruiting the available motors, potentially depleting the reservoir and affecting cytoskeletal transport. In this work we provide a theoretical framework in which to study, analytically and numerically, how motor density profiles and crowding along cytoskeletal filaments depend on the competition of motors for their binding sites. We propose two models in which finite processive motor proteins actively advance along cytoskeletal filaments and are continuously exchanged with the motor pool. We first look at homogeneous reservoirs and then examine the effects of free motor diffusion in the surrounding medium. We consider as a reference situation recent in vitro experimental setups of kinesin-8 motors binding and moving along microtubule filaments in a flow chamber. We investigate how the crowding of linear motor proteins moving on a filament can be regulated by the balance between supply (concentration of motor proteins in the flow chamber) and demand (total number of polymerized tubulin heterodimers). We present analytical results for the density profiles of bound motors and the reservoir depletion, and propose novel phase diagrams that present the formation of jams of motor proteins on the filament as a function of two tuneable experimental parameters: the motor protein concentration and the concentration of tubulins polymerized into cytoskeletal filaments. Extensive numerical simulations corroborate the analytical results for parameters in the experimental range and also address the effects of diffusion of motor proteins in the reservoir. We then propose experiments for validating our models and discuss how the ‘supply–demand’ effects can regulate motor traffic also in in vivo

Biochemical and functional characterization of an albumin protein belonging to the hemopexin superfamily from Lens culinaris seeds.

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Scarafoni, Alessio; Gualtieri, Elisa; Barbiroli, Alberto; Carpen, Aristodemo; Negri, Armando; Duranti, Marcello

2011-09-14

The present paper reports the purification and biochemical characterization of an albumin identified in mature lentil seeds with high sequence similarity to pea PA2. These proteins are found in many edible seeds and are considered potentially detrimental for human health due to the potential allergenicity and lectin-like activity. Thus, the description of their possible presence in food and the assessment of the molecular properties are relevant. The M(r), pI, and N-terminal sequence of this protein have been determined. The work included the study of (i) the binding properties to hemine to assess the presence of hemopexin structural domains and (ii) the binding properties of the protein to thiamin. In addition, the structural changes induced by heating have been evaluated by means of spectroscopic techniques. Denaturation temperature has also been determined. The present work provides new insights about the structural molecular features and the ligand-binding properties and dynamics of this kind of seed albumin.

Dissecting signaling and functions of adhesion G protein-coupled receptors

DEFF Research Database (Denmark)

Araç, Demet; Aust, Gabriela; Calebiro, Davide

2012-01-01

G protein-coupled receptors (GPCRs) comprise an expanded superfamily of receptors in the human genome. Adhesion class G protein-coupled receptors (adhesion-GPCRs) form the second largest class of GPCRs. Despite the abundance, size, molecular structure, and functions in facilitating cell and matrix...... contacts in a variety of organ systems, adhesion-GPCRs are by far the most poorly understood GPCR class. Adhesion-GPCRs possess a unique molecular structure, with extended N-termini containing various adhesion domains. In addition, many adhesion-GPCRs are autoproteolytically cleaved into an N......-terminal fragment (NTF, NT, α-subunit) and C-terminal fragment (CTF, CT, β-subunit) at a conserved GPCR autoproteolysis-inducing (GAIN) domain that contains a GPCR proteolysis site (GPS). These two features distinguish adhesion-GPCRs from other GPCR classes. Though active research on adhesion-GPCRs in diverse areas...

The tomato RLK superfamily: phylogeny and functional predictions about the role of the LRRII-RLK subfamily in antiviral defense.

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Sakamoto, Tetsu; Deguchi, Michihito; Brustolini, Otávio J B; Santos, Anésia A; Silva, Fabyano F; Fontes, Elizabeth P B

2012-12-02

Receptor-like kinases (RLKs) play key roles during development and in responses to the environment. Despite the relevance of the RLK family and the completion of the tomato genome sequencing, the tomato RLK family has not yet been characterized, and a framework for functional predictions of the members of the family is lacking. To generate a complete list of all the members of the tomato RLK family, we performed a phylogenetic analysis using the Arabidopsis family as a template. A total of 647 RLKs were identified in the tomato genome, which were organized into the same subfamily clades as Arabidopsis RLKs. Only eight of 58 RLK subfamilies exhibited specific expansion/reduction compared to their Arabidopsis counterparts. We also characterized the LRRII-RLK family by phylogeny, genomic analysis, expression profile and interaction with the virulence factor from begomoviruses, the nuclear shuttle protein (NSP). The LRRII subfamily members from tomato and Arabidopsis were highly conserved in both sequence and structure. Nevertheless, the majority of the orthologous pairs did not display similar conservation in the gene expression profile, indicating that these orthologs may have diverged in function after speciation. Based on the fact that members of the Arabidopsis LRRII subfamily (AtNIK1, AtNIK2 and AtNIK3) interact with the begomovirus nuclear shuttle protein (NSP), we examined whether the tomato orthologs of NIK, BAK1 and NsAK genes interact with NSP of Tomato Yellow Spot Virus (ToYSV). The tomato orthologs of NSP interactors, SlNIKs and SlNsAK, interacted specifically with NSP in yeast and displayed an expression pattern consistent with the pattern of geminivirus infection. In addition to suggesting a functional analogy between these phylogenetically classified orthologs, these results expand our previous observation that NSP-NIK interactions are neither virus-specific nor host-specific. The tomato RLK superfamily is made-up of 647 proteins that form a

The tomato RLK superfamily: phylogeny and functional predictions about the role of the LRRII-RLK subfamily in antiviral defense

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Sakamoto Tetsu

2012-12-01

superfamily is made-up of 647 proteins that form a monophyletic tree with the Arabidopsis RLKs and is divided into 58 subfamilies. Few subfamilies have undergone expansion/reduction, and only six proteins were lineage-specific. Therefore, the tomato RLK family shares functional and structural conservation with Arabidopsis. For the LRRII-RLK members SlNIK1 and SlNIK3, we observed functions analogous to those of their Arabidopsis counterparts with respect to protein-protein interactions and similar expression profiles, which predominated in tissues that support high efficiency of begomovirus infection. Therefore, NIK-mediated antiviral signaling is also likely to operate in tomato, suggesting that tomato NIKs may be good targets for engineering resistance against tomato-infecting begomoviruses.

Efficient Feature Selection and Classification of Protein Sequence Data in Bioinformatics

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Faye, Ibrahima; Samir, Brahim Belhaouari; Md Said, Abas

2014-01-01

Bioinformatics has been an emerging area of research for the last three decades. The ultimate aims of bioinformatics were to store and manage the biological data, and develop and analyze computational tools to enhance their understanding. The size of data accumulated under various sequencing projects is increasing exponentially, which presents difficulties for the experimental methods. To reduce the gap between newly sequenced protein and proteins with known functions, many computational techniques involving classification and clustering algorithms were proposed in the past. The classification of protein sequences into existing superfamilies is helpful in predicting the structure and function of large amount of newly discovered proteins. The existing classification results are unsatisfactory due to a huge size of features obtained through various feature encoding methods. In this work, a statistical metric-based feature selection technique has been proposed in order to reduce the size of the extracted feature vector. The proposed method of protein classification shows significant improvement in terms of performance measure metrics: accuracy, sensitivity, specificity, recall, F-measure, and so forth. PMID:25045727

Identification of KIF3A as a Novel Candidate Gene for Childhood Asthma Using RNA Expression and Population Allelic Frequencies Differences

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Butsch Kovacic, Melinda; Biagini Myers, Jocelyn M.; Wang, Ning; Martin, Lisa J.; Lindsey, Mark; Ericksen, Mark B.; He, Hua; Patterson, Tia L.; Baye, Tesfaye M.; Torgerson, Dara; Roth, Lindsey A.; Gupta, Jayanta; Sivaprasad, Umasundari; Gibson, Aaron M.; Tsoras, Anna M.; Hu, Donglei; Eng, Celeste; Chapela, Rocío; Rodríguez-Santana, José R.; Rodríguez-Cintrón, William; Avila, Pedro C.; Beckman, Kenneth; Seibold, Max A.; Gignoux, Chris; Musaad, Salma M.; Chen, Weiguo; Burchard, Esteban González; Khurana Hershey, Gurjit K.

2011-01-01

Background Asthma is a chronic inflammatory disease with a strong genetic predisposition. A major challenge for candidate gene association studies in asthma is the selection of biologically relevant genes. Methodology/Principal Findings Using epithelial RNA expression arrays, HapMap allele frequency variation, and the literature, we identified six possible candidate susceptibility genes for childhood asthma including ADCY2, DNAH5, KIF3A, PDE4B, PLAU, SPRR2B. To evaluate these genes, we compared the genotypes of 194 predominantly tagging SNPs in 790 asthmatic, allergic and non-allergic children. We found that SNPs in all six genes were nominally associated with asthma (pasthma (OR = 2.3, pasthma population attributable risk of 18.5%. The association between KIF3A rs7737031 and asthma was validated in 3 independent populations, further substantiating the validity of our gene selection approach. Conclusions/Significance Our study demonstrates that KIF3A, a member of the kinesin superfamily of microtubule associated motors that are important in the transport of protein complexes within cilia, is a novel candidate gene for childhood asthma. Polymorphisms in KIF3A may in part be responsible for poor mucus and/or allergen clearance from the airways. Furthermore, our study provides a promising framework for the identification and evaluation of novel candidate susceptibility genes. PMID:21912604

Computational Identification of the Paralogs and Orthologs of Human Cytochrome P450 Superfamily and the Implication in Drug Discovery

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Shu-Ting Pan

2016-06-01

Full Text Available The human cytochrome P450 (CYP superfamily consisting of 57 functional genes is the most important group of Phase I drug metabolizing enzymes that oxidize a large number of xenobiotics and endogenous compounds, including therapeutic drugs and environmental toxicants. The CYP superfamily has been shown to expand itself through gene duplication, and some of them become pseudogenes due to gene mutations. Orthologs and paralogs are homologous genes resulting from speciation or duplication, respectively. To explore the evolutionary and functional relationships of human CYPs, we conducted this bioinformatic study to identify their corresponding paralogs, homologs, and orthologs. The functional implications and implications in drug discovery and evolutionary biology were then discussed. GeneCards and Ensembl were used to identify the paralogs of human CYPs. We have used a panel of online databases to identify the orthologs of human CYP genes: NCBI, Ensembl Compara, GeneCards, OMA (“Orthologous MAtrix” Browser, PATHER, TreeFam, EggNOG, and Roundup. The results show that each human CYP has various numbers of paralogs and orthologs using GeneCards and Ensembl. For example, the paralogs of CYP2A6 include CYP2A7, 2A13, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2F1, 2J2, 2R1, 2S1, 2U1, and 2W1; CYP11A1 has 6 paralogs including CYP11B1, 11B2, 24A1, 27A1, 27B1, and 27C1; CYP51A1 has only three paralogs: CYP26A1, 26B1, and 26C1; while CYP20A1 has no paralog. The majority of human CYPs are well conserved from plants, amphibians, fishes, or mammals to humans due to their important functions in physiology and xenobiotic disposition. The data from different approaches are also cross-validated and validated when experimental data are available. These findings facilitate our understanding of the evolutionary relationships and functional implications of the human CYP superfamily in drug discovery.

Proteomics investigation reveals cell death-associated proteins of basidiomycete fungus Trametes versicolor treated with Ferruginol.

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Chen, Yu-Han; Yeh, Ting-Feng; Chu, Fang-Hua; Hsu, Fu-Lan; Chang, Shang-Tzen

2015-01-14

Ferruginol has antifungal activity against wood-rot fungi (basidiomycetes). However, specific research on the antifungal mechanisms of ferruginol is scarce. Two-dimensional gel electrophoresis and fluorescent image analysis were employed to evaluate the differential protein expression of wood-rot fungus Trametes versicolor treated with or without ferruginol. Results from protein identification of tryptic peptides via liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) analyses revealed 17 protein assignments with differential expression. Downregulation of cytoskeleton β-tubulin 3 indicates that ferruginol has potential to be used as a microtubule-disrupting agent. Downregulation of major facilitator superfamily (MFS)–multiple drug resistance (MDR) transporter and peroxiredoxin TSA1 were observed, suggesting reduction in self-defensive capabilities of T. versicolor. In addition, the proteins involved in polypeptide sorting and DNA repair were also downregulated, while heat shock proteins and autophagy-related protein 7 were upregulated. These observations reveal that such cellular dysfunction and damage caused by ferruginol lead to growth inhibition and autophagic cell death of fungi.

Light activated bionanodevices

CSIR Research Space (South Africa)

Sparrow, RW

2006-02-27

Full Text Available production and Kinesin motor protein movement. It has been designed in a modular concept with three sections: energy trapping (light harvesting) and transfer; energy conversion to produce ATP; and mechanical translation. The potential applications of such a...

Structure of the N-terminal domain of the protein Expansion: an ‘Expansion’ to the Smad MH2 fold

International Nuclear Information System (INIS)

Beich-Frandsen, Mads; Aragón, Eric; Llimargas, Marta; Benach, Jordi; Riera, Antoni; Pous, Joan; Macias, Maria J.

2015-01-01

Expansion is a modular protein that is conserved in protostomes. The first structure of the N-terminal domain of Expansion has been determined at 1.6 Å resolution and the new Nα-MH2 domain was found to belong to the Smad/FHA superfamily of structures. Gene-expression changes observed in Drosophila embryos after inducing the transcription factor Tramtrack led to the identification of the protein Expansion. Expansion contains an N-terminal domain similar in sequence to the MH2 domain characteristic of Smad proteins, which are the central mediators of the effects of the TGF-β signalling pathway. Apart from Smads and Expansion, no other type of protein belonging to the known kingdoms of life contains MH2 domains. To compare the Expansion and Smad MH2 domains, the crystal structure of the Expansion domain was determined at 1.6 Å resolution, the first structure of a non-Smad MH2 domain to be characterized to date. The structure displays the main features of the canonical MH2 fold with two main differences: the addition of an α-helical region and the remodelling of a protein-interaction site that is conserved in the MH2 domain of Smads. Owing to these differences, to the new domain was referred to as Nα-MH2. Despite the presence of the Nα-MH2 domain, Expansion does not participate in TGF-β signalling; instead, it is required for other activities specific to the protostome phyla. Based on the structural similarities to the MH2 fold, it is proposed that the Nα-MH2 domain should be classified as a new member of the Smad/FHA superfamily

Structure of the N-terminal domain of the protein Expansion: an ‘Expansion’ to the Smad MH2 fold

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Beich-Frandsen, Mads; Aragón, Eric [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Llimargas, Marta [Institut de Biologia Molecular de Barcelona, IBMB–CSIC, Baldiri Reixac 10, 08028 Barcelona (Spain); Benach, Jordi [ALBA Synchrotron, BP 1413, km 3.3, Cerdanyola del Vallès (Spain); Riera, Antoni [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Universitat de Barcelona, Martí i Franqués 1-11, 08028 Barcelona (Spain); Pous, Joan [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Platform of Crystallography IBMB–CSIC, Baldiri Reixac 10, 08028 Barcelona (Spain); Macias, Maria J., E-mail: maria.macias@irbbarcelona.org [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Catalan Institution for Research and Advanced Studies (ICREA), Passeig Lluís Companys 23, 08010 Barcelona (Spain)

2015-04-01

Expansion is a modular protein that is conserved in protostomes. The first structure of the N-terminal domain of Expansion has been determined at 1.6 Å resolution and the new Nα-MH2 domain was found to belong to the Smad/FHA superfamily of structures. Gene-expression changes observed in Drosophila embryos after inducing the transcription factor Tramtrack led to the identification of the protein Expansion. Expansion contains an N-terminal domain similar in sequence to the MH2 domain characteristic of Smad proteins, which are the central mediators of the effects of the TGF-β signalling pathway. Apart from Smads and Expansion, no other type of protein belonging to the known kingdoms of life contains MH2 domains. To compare the Expansion and Smad MH2 domains, the crystal structure of the Expansion domain was determined at 1.6 Å resolution, the first structure of a non-Smad MH2 domain to be characterized to date. The structure displays the main features of the canonical MH2 fold with two main differences: the addition of an α-helical region and the remodelling of a protein-interaction site that is conserved in the MH2 domain of Smads. Owing to these differences, to the new domain was referred to as Nα-MH2. Despite the presence of the Nα-MH2 domain, Expansion does not participate in TGF-β signalling; instead, it is required for other activities specific to the protostome phyla. Based on the structural similarities to the MH2 fold, it is proposed that the Nα-MH2 domain should be classified as a new member of the Smad/FHA superfamily.

Cellular and molecular biology of orphan G protein-coupled receptors.

Science.gov (United States)

Oh, Da Young; Kim, Kyungjin; Kwon, Hyuk Bang; Seong, Jae Young

2006-01-01

The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of membrane-spanning proteins. It plays a variety of roles in pathophysiological processes by transmitting extracellular signals to cells via heterotrimeric G proteins. Completion of the human genome project revealed the presence of approximately 168 genes encoding established nonsensory GPCRs, as well as 207 genes predicted to encode novel GPCRs for which the natural ligands remained to be identified, the so-called orphan GPCRs. Eighty-six of these orphans have now been paired to novel or previously known molecules, and 121 remain to be deorphaned. A better understanding of the GPCR structures and classification; knowledge of the receptor activation mechanism, either dependent on or independent of an agonist; increased understanding of the control of GPCR-mediated signal transduction; and development of appropriate ligand screening systems may improve the probability of discovering novel ligands for the remaining orphan GPCRs.

Conventional kinesin KIF5B mediates adiponectin secretion in 3T3-L1 adipocytes

Energy Technology Data Exchange (ETDEWEB)

Cui, Ju, E-mail: juzi.cui@gmail.com [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China); Pang, Jing; Lin, Ya-Jun; Jiang, Ping; Gong, Huan [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China); Wang, Zai [Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing (China); Li, Jian; Cai, Jian-Ping [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China); Huang, Jian-Dong, E-mail: jdhuang@hku.hk [School of Biomedical Sciences and Shenzhen Institute of Research and Innovation, The University of Hong Kong, Pokfulam (Hong Kong); The Centre for Synthetic Biology Engineering Research, Shenzhen Institutes of Advanced Technology, Shenzhen (China); Zhang, Tie-Mei, E-mail: tmzhang126@126.com [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China)

2016-08-05

Insulin stimulates adiponectin secretion and glucose transporter type 4 (GLUT4) translocation in adipocyte to regulate metabolism homeostasis. Similar to GLUT4 translocation, intracellular trafficking and release of adiponectin in adipocytes relies on the trans-Golgi network and endosomal system. Recent studies show that the heavy chain of conventional kinesin (KIF5B) mediates GLUT4 translocation in murine 3T3-L1 adipocytes, however, the motor machinery involved in mediating intracellular trafficking and release of adiponectin is unknown. Here, we examined the role of KIF5B in the regulation of adiponectin secretion. The KIF5B level was up-regulated during 3T3-L1 adipogenesis. This increase in cytosolic KIF5B was synchronized with the induction of adiponectin. Endogenous KIF5B and adiponectin were partially colocalized at the peri-nuclear and cytosolic regions. In addition, adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. Knockdown of KIF5B resulted in a marked inhibition of adiponectin secretion and overexpression of KIF5B enhanced adiponectin release, whereas leptin secretion was not affected by changes in KIF5B expression. These data suggest that the secretion of adiponectin, but not leptin, is dependent on functional KIF5B. - Highlights: • The KIF5B level was up regulated during 3T3-L1 adipogenesis. • Endogenous KIF5B and adiponectin were partially colicalized. • Adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. • The secretion of adiponectin, but not leptin, is dependent on functional KIF5B.

Conventional kinesin KIF5B mediates adiponectin secretion in 3T3-L1 adipocytes

International Nuclear Information System (INIS)

Cui, Ju; Pang, Jing; Lin, Ya-Jun; Jiang, Ping; Gong, Huan; Wang, Zai; Li, Jian; Cai, Jian-Ping; Huang, Jian-Dong; Zhang, Tie-Mei

2016-01-01

Insulin stimulates adiponectin secretion and glucose transporter type 4 (GLUT4) translocation in adipocyte to regulate metabolism homeostasis. Similar to GLUT4 translocation, intracellular trafficking and release of adiponectin in adipocytes relies on the trans-Golgi network and endosomal system. Recent studies show that the heavy chain of conventional kinesin (KIF5B) mediates GLUT4 translocation in murine 3T3-L1 adipocytes, however, the motor machinery involved in mediating intracellular trafficking and release of adiponectin is unknown. Here, we examined the role of KIF5B in the regulation of adiponectin secretion. The KIF5B level was up-regulated during 3T3-L1 adipogenesis. This increase in cytosolic KIF5B was synchronized with the induction of adiponectin. Endogenous KIF5B and adiponectin were partially colocalized at the peri-nuclear and cytosolic regions. In addition, adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. Knockdown of KIF5B resulted in a marked inhibition of adiponectin secretion and overexpression of KIF5B enhanced adiponectin release, whereas leptin secretion was not affected by changes in KIF5B expression. These data suggest that the secretion of adiponectin, but not leptin, is dependent on functional KIF5B. - Highlights: • The KIF5B level was up regulated during 3T3-L1 adipogenesis. • Endogenous KIF5B and adiponectin were partially colicalized. • Adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. • The secretion of adiponectin, but not leptin, is dependent on functional KIF5B.

Multiple Receptor Subtypes for the CGRP Super-Family

Directory of Open Access Journals (Sweden)

R. Quirion

2001-01-01

Full Text Available Molecular evidence for the existence of multiple receptors for CGRP has been rather difficult to obtain. Over 10 years after suggesting the existence of at least two classes (CGRP1 and CGRP2 of CGRP receptors on the basis of pharmacological data[1], molecular data on the CGRP2 receptor subtype are still lacking as well as potent and selective antagonists. The situation is somewhat different for the functional CGRP1 subtype which is likely composed of diverse subunits CRLR, RAMP1 and possibly RCP[2]. Moreover, BIBN 4096BS was recently reported as the first nonpeptide highly potent CGRP1 receptor antagonist[3]. However, in situ hybridization and receptor autoradiographic data have clearly shown the existence of major mismatches (e.g., cerebellum between the discrete localization of CRLR, RAMP1, and specific CGRP binding sites supporting the existence of CGRP receptor subtypes. Functional studies have also provided evidence in that regard (for a recent review: [4]. Accordingly, additional studies aiming at cloning additional CGRP receptors are certainly warranted. Similarly, recent evidence from various laboratories including ours suggests the existence of more than one class (CRLR and RAMP2 of adrenomedullin receptors at least in the rat brain. In contrast, most evidence suggests the existence of a single class of amylin receptors. In brief, it appears that multiple receptors or receptor complexes do exist for CGRP and related peptides but their composition is apparently unique among the GPCR super-family and additional data are needed to fully establish the molecular organization of each subtype. Supported by CIHR of Canada.

The Origin and Early Evolution of Membrane Proteins

Science.gov (United States)

Pohorille, Andrew; Schweighofter, Karl; Wilson, Michael A.

2006-01-01

The origin and early evolution of membrane proteins, and in particular ion channels, are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Prokarya, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

Protein (Cyanobacteria): 493030290 [PGDBj - Ortholog DB

Lifescience Database Archive (English)

Full Text Available oxin T-superfamily Coleofasciculus chthonoplastes MGGDNSKKPVLGLMNKGGFCCSVIGEEGGFCCSVIGEEGGFCCSVIGEEGGFCCSVIGEEGGFCCSVIGEEGGFCCSVIGEEGGFCCSVIGEEGGFCCSVIGEEGGFCCSVIGEEGGFCCSVISAIADFSP

Two Major Facilitator Superfamily Sugar Transporters from Trichoderma reesei and Their Roles in Induction of Cellulase Biosynthesis*

Science.gov (United States)

Zhang, Weixin; Kou, Yanbo; Xu, Jintao; Cao, Yanli; Zhao, Guolei; Shao, Jing; Wang, Hai; Wang, Zhixing; Bao, Xiaoming; Chen, Guanjun; Liu, Weifeng

2013-01-01

Proper perception of the extracellular insoluble cellulose is key to initiating the rapid synthesis of cellulases by cellulolytic Trichoderma reesei. Uptake of soluble oligosaccharides derived from cellulose hydrolysis represents a potential point of control in the induced cascade. In this study, we identified a major facilitator superfamily sugar transporter Stp1 capable of transporting cellobiose by reconstructing a cellobiose assimilation system in Saccharomyces cerevisiae. The absence of Stp1 in T. reesei resulted in differential cellulolytic response to Avicel versus cellobiose. Transcriptional profiling revealed a different expression profile in the Δstp1 strain from that of wild-type strain in response to Avicel and demonstrated that Stp1 somehow repressed induction of the bulk of major cellulase and hemicellulose genes. Two other putative major facilitator superfamily sugar transporters were, however, up-regulated in the profiling. Deletion of one of them identified Crt1 that was required for growth and enzymatic activity on cellulose or lactose, but was not required for growth or hemicellulase activity on xylan. The essential role of Crt1 in cellulase induction did not seem to rely on its transporting activity because the overall uptake of cellobiose or sophorose by T. reesei was not compromised in the absence of Crt1. Phylogenetic analysis revealed that orthologs of Crt1 exist in the genomes of many filamentous ascomycete fungi capable of degrading cellulose. These data thus shed new light on the mechanism by which T. reesei senses and transmits the cellulose signal and offers potential strategies for strain improvement. PMID:24085297

Benzoate-mediated changes on expression profile of soluble proteins in Serratia sp. DS001.

Science.gov (United States)

Pandeeti, E V P; Chinnaboina, M R; Siddavattam, D

2009-05-01

To assess differences in protein expression profile associated with shift in carbon source from succinate to benzoate in Serratia sp. DS001 using a proteomics approach. A basic proteome map was generated for the soluble proteins extracted from Serratia sp. DS001 grown in succinate and benzoate. The differently and differentially expressed proteins were identified using ImageMaster 2D Platinum software (GE Healthcare). The identity of the proteins was determined by employing MS or MS/MS. Important enzymes such as Catechol 1,2 dioxygenase and transcriptional regulators that belong to the LysR superfamily were identified. Nearly 70 proteins were found to be differentially expressed when benzoate was used as carbon source. Based on the protein identity and degradation products generated from benzoate it is found that ortho pathway is operational in Serratia sp. DS001. Expression profile of the soluble proteins associated with shift in carbon source was mapped. The study also elucidates degradation pathway of benzoate in Serratia sp. DS001 by correlating the proteomics data with the catabolites of benzoate.

Cytokinesis defect in BY-2 cells caused by ATP-competitive kinase inhibitors.

Science.gov (United States)

Kozgunova, Elena; Higashiyama, Tetsuya; Kurihara, Daisuke

2016-10-02

Cytokinesis is last but not least in cell division as it completes the formation of the two cells. The main role in cell plate orientation and expansion have been assigned to microtubules and kinesin proteins. However, recently we reported severe cytokinesis defect in BY-2 cells not accompanied by changes in microtubules dynamics. Here we also confirmed that distribution of kinesin NACK1 is not the cause of cytokinesis defect. We further explored inhibition of the cell plate expansion by ATP-competitive inhibitors. Two different inhibitors, 5-Iodotubercidin and ML-7 resulted in a very similar phenotype, which indicates that they target same protein cascade. Interestingly, in our previous study we showed that 5-Iodotubercidin treatment affects concentration of actin filaments on the cell plate, while ML-7 is inhibitor of myosin light chain kinase. Although not directly, it indicates importance of actomyosin complex in plant cytokinesis.

COGcollator: a web server for analysis of distant relationships between homologous protein families.

Science.gov (United States)

Dibrova, Daria V; Konovalov, Kirill A; Perekhvatov, Vadim V; Skulachev, Konstantin V; Mulkidjanian, Armen Y

2017-11-29

The Clusters of Orthologous Groups (COGs) of proteins systematize evolutionary related proteins into specific groups with similar functions. However, the available databases do not provide means to assess the extent of similarity between the COGs. We intended to provide a method for identification and visualization of evolutionary relationships between the COGs, as well as a respective web server. Here we introduce the COGcollator, a web tool for identification of evolutionarily related COGs and their further analysis. We demonstrate the utility of this tool by identifying the COGs that contain distant homologs of (i) the catalytic subunit of bacterial rotary membrane ATP synthases and (ii) the DNA/RNA helicases of the superfamily 1. This article was reviewed by Drs. Igor N. Berezovsky, Igor Zhulin and Yuri Wolf.

Structural organization of the genes for rat von Ebner's gland proteins 1 and 2 reveals their close relationship to lipocalins.

Science.gov (United States)

Kock, K; Ahlers, C; Schmale, H

1994-05-01

The rat von Ebner's gland protein 1 (VEGP 1) is a secretory protein, which is abundantly expressed in the small acinar von Ebner's salivary glands of the tongue. Based on the primary structure of this protein we have previously suggested that it is a member of the lipocalin superfamily of lipophilic-ligand carrier proteins. Although the physiological role of VEGP 1 is not clear, it might be involved in sensory or protective functions in the taste epithelium. Here, we report the purification of VEGP 1 and of a closely related secretory polypeptide, VEGP 2, the isolation of a cDNA clone encoding VEGP 2, and the isolation and structural characterization of the genes for both proteins. Protein purification by gel-filtration and anion-exchange chromatography using Mono Q revealed the presence of two different immunoreactive VEGP species. N-terminal sequence determination of peptide fragments isolated after protease Asp-N digestion allowed the identification of a new VEGP, named VEGP 2, in addition to the previously characterized VEGP 1. The complete VEGP 2 sequence was deduced from a cDNA clone isolated from a von Ebner's gland cDNA library. The VEGP 2 cDNA encodes a protein of 177 amino acids and is 94% identical to VEGP 1. DNA sequence analysis of the rat VEGP 1 and 2 genes isolated from rat genomic libraries revealed that both span about 4.5 kb and contain seven exons. The VEGP 1 and 2 genes are non-allelic distinct genes in the rat genome and probably arose by gene duplication. The high degree of nucleotide sequence identity in introns A-C (94-100%) points to a recent gene conversion event that included the 5' part of the genes. The genomic organization of the rat VEGP genes closely resembles that found in other lipocalins such as beta-lactoglobulin, mouse urinary proteins (MUPs) and prostaglandin D synthase, and therefore provides clear evidence that VEGPs belong to this superfamily of proteins.

Importance of anisotropy in detachment rates for force production and cargo transport by a team of motor proteins.

Science.gov (United States)

Takshak, Anjneya; Kunwar, Ambarish

2016-05-01

Many cellular processes are driven by collective forces generated by a team consisting of multiple molecular motor proteins. One aspect that has received less attention is the detachment rate of molecular motors under mechanical force/load. While detachment rate of kinesin motors measured under backward force increases rapidly for forces beyond stall-force; this scenario is just reversed for non-yeast dynein motors where detachment rate from microtubule decreases, exhibiting a catch-bond type behavior. It has been shown recently that yeast dynein responds anisotropically to applied load, i.e. detachment rates are different under forward and backward pulling. Here, we use computational modeling to show that these anisotropic detachment rates might help yeast dynein motors to improve their collective force generation in the absence of catch-bond behavior. We further show that the travel distance of cargos would be longer if detachment rates are anisotropic. Our results suggest that anisotropic detachment rates could be an alternative strategy for motors to improve the transport properties and force production by the team. © 2016 The Protein Society.

Cysteine-dependent immune regulation by TRX and MIF/GIF family proteins.

Science.gov (United States)

Kondo, Norihiko; Ishii, Yasuyuki; Son, Aoi; Sakakura-Nishiyama, Junko; Kwon, Yong-Won; Tanito, Masaki; Nishinaka, Yumiko; Matsuo, Yoshiyuki; Nakayama, Toshinori; Taniguchi, Masaru; Yodoi, Junji

2004-03-29

Thioredoxin (TRX) superfamily proteins that contain a conserved redox-active site -Cys-Xa.a.-Xa.a.-Cys- includes proinflammatory cytokine, macrophage migration inhibiting factor (MIF) and the immune regulatory cytokine, glycosylation inhibiting factor (GIF) in which Cys-60 is cysteinylated. In this report, we have analyzed the functional interaction between TRX and MIF/GIF. The stable Jurkat T cell line transfected with human TRX gene (TRX-transfectant) was highly resistant to hydrogen peroxide-induced apoptosis, but not the cell line transfected with vector (mock-transfectant). The expression level of MIF/GIF protein of TRX-transfectant was lower than that of mock-transfectant. Conversely, the expression level of intracellular TRX protein in CD4(+)-T cells derived from MIF -/- mice were significantly higher than that from background BALB/c mice. These findings collectively suggest that oxidative stress-induced apoptosis on T lymphocytes might be protected by the reciprocal regulation of TRX and MIF/GIF expression.

Annotating Enzymes of Uncertain Function: The Deacylation of d-Amino Acids by Members of the Amidohydrolase Superfamily

Energy Technology Data Exchange (ETDEWEB)

Cummings, J.; Fedorov, A; Xu, C; Brown, S; Fedorov, E; Babbitt, P; Almo, S; Raushel, F

2009-01-01

The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxidans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22-34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, N-succinyl-d-Xaa, and l-Xaa-d-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl-substituted derivatives of d-glutamate. The best substrates for this enzyme are N-formyl-d-glutamate (k{sub cat}/K{sub m} = 5.8 x 10{sup 6} M{sup -1} s{sup -1}), N-acetyl-d-glutamate (k{sub cat}/K{sub m} = 5.2 x 10{sup 6} M{sup -1} s{sup -1}), and l-methionine-d-glutamate (k{sub cat}/K{sub m} = 3.4 x 10{sup 5} M{sup -1} s{sup -1}). Gox1177 and Sco4986 preferentially hydrolyze N-acyl-substituted derivatives of hydrophobic d-amino acids. The best substrates for Gox1177 are N-acetyl-d-leucine (k{sub cat}/K{sub m} = 3.2 x 104 M{sup -1} s-1), N-acetyl-d-tryptophan (kcat/Km = 4.1 x 104 M-1 s-1), and l-tyrosine-d-leucine (kcat/Km = 1.5 x 104 M-1 s-1). A fourth protein, Bb2785 from B. bronchiseptica, did not have d-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-d-phenylalanine and N-acetyl-d-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the d-glutamate moiety of the inhibitor is ion-paired to Arg-295, while the {alpha}-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein. Bioinformatic analyses of an additional {approx}250

Role for ribosome-associated complex and stress-seventy subfamily B (RAC-Ssb) in integral membrane protein translation.

Science.gov (United States)

Acosta-Sampson, Ligia; Döring, Kristina; Lin, Yuping; Yu, Vivian Y; Bukau, Bernd; Kramer, Günter; Cate, Jamie H D

2017-12-01

Targeting of most integral membrane proteins to the endoplasmic reticulum is controlled by the signal recognition particle, which recognizes a hydrophobic signal sequence near the protein N terminus. Proper folding of these proteins is monitored by the unfolded protein response and involves protein degradation pathways to ensure quality control. Here, we identify a new pathway for quality control of major facilitator superfamily transporters that occurs before the first transmembrane helix, the signal sequence recognized by the signal recognition particle, is made by the ribosome. Increased rates of translation elongation of the N-terminal sequence of these integral membrane proteins can divert the nascent protein chains to the ribosome-associated complex and stress-seventy subfamily B chaperones. We also show that quality control of integral membrane proteins by ribosome-associated complex-stress-seventy subfamily B couples translation rate to the unfolded protein response, which has implications for understanding mechanisms underlying human disease and protein production in biotechnology. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

NCX-DB: a unified resource for integrative analysis of the sodium calcium exchanger super-family.

Science.gov (United States)

Bode, Katrin; O'Halloran, Damien M

2018-04-13

Na + /Ca 2+ exchangers are low-affinity high-capacity transporters that mediate Ca 2+ extrusion by coupling Ca 2+ efflux to the influx of Na + ions. The Na + /Ca 2+ exchangers form a super-family comprised of three branches each differing in ion-substrate selectivity: Na + /Ca 2+ exchangers (NCX), Na + /Ca 2+ /K + exchangers, and Ca 2+ /cation exchangers. Their primary function is to maintain Ca 2+ homeostasis and play a particularly important role in excitable cells that experience transient Ca 2+ fluxes. Research into the role and activity of Na + /Ca 2+ exchangers has focused extensively on the cardio-vascular system, however, growing evidence suggests that Na + /Ca 2+ exchangers play a key role in neuronal processes such as memory formation, learning, oligodendrocyte differentiation, neuroprotection during brain ischemia and axon guidance. They have also been implicated in pathologies such as Alzheimer's disease, Parkinson's disease, Multiple Sclerosis and Epilepsy, however, a clear understanding of their mechanism during disease is lacking. To date, there has never been a central resource or database for Na + /Ca 2+ exchangers. With clear disease relevance and ever-increasing research on Na + /Ca 2+ exchangers from both model and non-model species, a database that unifies the data on Na + /Ca 2+ exchangers is needed for future research. NCX-DB is a publicly available database with a web interface that enables users to explore various Na + /Ca 2+ exchangers, perform cross-species sequence comparison, identify new exchangers, and stay-up to date with recent literature. NCX-DB is available on the web via an interactive user interface with an intuitive design, which is applicable for the identification and comparison of Na + /Ca 2+ exchanger proteins across diverse species.

Photocontrol of the mitotic kinesin Eg5 using a novel S-trityl-L-cysteine analogue as a photochromic inhibitor.

Science.gov (United States)

Ishikawa, Kumiko; Tohyama, Kanako; Mitsuhashi, Shinya; Maruta, Shinsaku

2014-04-01

Because the mitotic kinesin Eg5 is essential for the formation of bipolar spindles during eukaryotic cell division, it has been considered as a potential target for cancer treatment. A number of specific and potent inhibitors of Eg5 are known. S-trityl-L-cysteine is one of the inhibitors of Eg5 whose molecular mechanism of inhibition was well studied. The trityl group of S-trityl-L-cysteine was shown to be a key moiety required for potent inhibition. In this study, we synthesized a novel photochromic S-trityl-L-cysteine analogue, 4-(N-(2-(N-acetylcysteine-S-yl) acetyl) amino)-4'- (N-(2-(N-(triphenylmethyl)amino)acetyl)amino)azobenzene (ACTAB), composed of a trityl group, azobenzene and N-acetyl-L-cysteine, which exhibits cis-trans photoisomerization in order to photocontrol the function of Eg5. ACTAB exhibited cis-trans photoisomerization upon alternating irradiation at two different wavelengths in the visible range, 400 and 480 nm. ACTAB induced reversible changes in the inhibitory activity of ATPase and motor activities correlating with the cis-trans photoisomerization. Compared with cis-ACTAB, trans-ACTAB reduced ATPase activity and microtubule gliding velocity more significantly. These results suggest that ACTAB could be used as photochromic inhibitor of Eg5 to achieve photocontrol of living cells.

Structural and Functional Analysis of VQ Motif-Containing Proteins in Arabidopsis as Interacting Proteins of WRKY Transcription Factors1[W][OA

Science.gov (United States)

Cheng, Yuan; Zhou, Yuan; Yang, Yan; Chi, Ying-Jun; Zhou, Jie; Chen, Jian-Ye; Wang, Fei; Fan, Baofang; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan; Chen, Zhixiang

2012-01-01

WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Recently, several groups have reported that proteins containing a short VQ (FxxxVQxLTG) motif interact with WRKY proteins. We have recently discovered that two VQ proteins from Arabidopsis (Arabidopsis thaliana), SIGMA FACTOR-INTERACTING PROTEIN1 and SIGMA FACTOR-INTERACTING PROTEIN2, act as coactivators of WRKY33 in plant defense by specifically recognizing the C-terminal WRKY domain and stimulating the DNA-binding activity of WRKY33. In this study, we have analyzed the entire family of 34 structurally divergent VQ proteins from Arabidopsis. Yeast (Saccharomyces cerevisiae) two-hybrid assays showed that Arabidopsis VQ proteins interacted specifically with the C-terminal WRKY domains of group I and the sole WRKY domains of group IIc WRKY proteins. Using site-directed mutagenesis, we identified structural features of these two closely related groups of WRKY domains that are critical for interaction with VQ proteins. Quantitative reverse transcription polymerase chain reaction revealed that expression of a majority of Arabidopsis VQ genes was responsive to pathogen infection and salicylic acid treatment. Functional analysis using both knockout mutants and overexpression lines revealed strong phenotypes in growth, development, and susceptibility to pathogen infection. Altered phenotypes were substantially enhanced through cooverexpression of genes encoding interacting VQ and WRKY proteins. These findings indicate that VQ proteins play an important role in plant growth, development, and response to environmental conditions, most likely by acting as cofactors of group I and IIc WRKY transcription factors. PMID:22535423

Mammalian O-mannosylation of Cadherins and Plexins is Independent of Protein O-mannosyltransferase 1 and 2

DEFF Research Database (Denmark)

Larsen, Ida Signe Bohse; Narimatsu, Yoshiki; Joshi, Hiren Jitendra

2017-01-01

Protein O-mannosylation is found in yeast and metazoans and a family of conserved orthologous protein O-mannosyltransferases is believed to initiate this important post-translational modification. We recently discovered that the cadherin superfamily carries O-linked mannose (O-Man) glycans...... at highly conserved residues in specific extracellular cadherin domains, and it was suggested that the function of E-cadherin was dependent on the O-Man glycans. Deficiencies in enzymes catalyzing O-Man biosynthesis, including the two human protein O-mannosyltransferases, POMT1 and POMT2, underlie...... a subgroup of congenital muscular dystrophies (CMD) designated α-dystroglycanopathies, because deficient O-Man glycosylation of -dystroglycan disrupts laminin interaction with -dystroglycan and the extracellular matrix. In order to explore the functions of O-Man glycans on cadherins and protocadherins we...

Dynein Heavy Chain, Encoded by Two Genes in Agaricomycetes, Is Required for Nuclear Migration in Schizophyllum commune.

Directory of Open Access Journals (Sweden)

Melanie Brunsch

Full Text Available The white-rot fungus Schizophyllum commune (Agaricomycetes was used to study the cell biology of microtubular trafficking during mating interactions, when the two partners exchange nuclei, which are transported along microtubule tracks. For this transport activity, the motor protein dynein is required. In S. commune, the dynein heavy chain is encoded in two parts by two separate genes, dhc1 and dhc2. The N-terminal protein Dhc1 supplies the dimerization domain, while Dhc2 encodes the motor machinery and the microtubule binding domain. This split motor protein is unique to Basidiomycota, where three different sequence patterns suggest independent split events during evolution. To investigate the function of the dynein heavy chain, the gene dhc1 and the motor domain in dhc2 were deleted. Both resulting mutants were viable, but revealed phenotypes in hyphal growth morphology and mating behavior as well as in sexual development. Viability of strain Δdhc2 is due to the higher expression of kinesin-2 and kinesin-14, which was proven via RNA sequencing.

Suppression of expression of muscle-associated proteins by PPARα in brown adipose tissue

International Nuclear Information System (INIS)

Tong, Yuhong; Hara, Atsushi; Komatsu, Makiko; Tanaka, Naoki; Kamijo, Yuji; Gonzalez, Frank J.; Aoyama, Toshifumi

2005-01-01

Peroxisome proliferator-activated receptor α (PPARα) belongs to the steroid/nuclear receptor superfamily. Two-dimensional (2D) SDS-PAGE analysis of brown adipose tissue (BAT) unexpectedly revealed six spots that were present only in PPARα-null mice. Proteomic analysis indicated that these proteins were tropomyosin-1 α chain, tropomyosin β chain, myosin regulatory light chain 2, myosin light chain 3, and parvalbumin α. Analyses of mRNA have revealed that PPARα suppressed the genes encoding these proteins in a synchronous manner in adult wild-type mice. Histological and physiological analyses of BAT showed in adult wild-type mice, a marked suppression of BAT growth concurrent with a prominent decrease in lipolytic and thermogenesis activities. These results suggest that in adult mice, PPARα functions to suppress the expression of the proteins that may be involved in the architecture of BAT, and thus may function in keeping BAT in a quiescent state

Presence of Foraminifera of Superfamily Komokioidea (Order Astrorhizida) in Colombian deep Caribbean waters.

Science.gov (United States)

Tavera-Martínez, Laura; Marchant, Margarita

2017-10-20

Research regarding deep-sea benthic foraminifera in the Colombian Caribbean requires further development given the complete lack of information related to the different groups that constitute associations and the ecological functions they fulfill. For this purpose, a taxonomic description of Superfamily Komokioidea was composed from macrofauna samples from between 1,215 m and 3,179 m depth, obtained during the research cruise ANH-COL 4 and COL 5 carried out in 2014. Results showed foraminifera belonging to the three families: Komokiidae, Baculellidae, and Normaninidae, inclu-ding five genera (Lana, Komokia, Ipoa, Normaninam, and Catena) and five species (Lana neglecta, Komokia multiramosa, Normanina conferta, Ipoa fragila, and Catena piriformis). This study presents knowledge regarding deep-sea Colombian Caribbean benthic foraminifera, which to date have not been recorded from this region. Their depth distribution when compared with other studies from the Atlantic and Pacific, allows the expansion of taxonomic inventories and the characterization of biodiversity within poorly explored regions.

Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

Energy Technology Data Exchange (ETDEWEB)

Rosenthal, Cindy [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Mueller, Uwe [Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung mbH, Albert-Einstein-Strasse 15, D-12489 Berlin (Germany); Panjikar, Santosh [European Molecular Biology Laboratory Hamburg, Outstation Deutsches Elektronen-Synchrotron, Notkestrasse 85, D-22603 Hamburg (Germany); Sun, Lianli [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China); Ruppert, Martin [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Zhao, Yu [Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China); Stöckigt, Joachim [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China)

2006-12-01

Perakine reductase, a novel member of the aldo-keto reductase enzyme superfamily of higher plants, is involved in the biosynthesis of monoterpenoid indole alkaloids in the Indian medicinal plant Rauvolfia serpentina. The enzyme has been crystallized in C-centered orthorhombic space group and diffracts to 2.0 Å resolution. Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C222{sub 1} and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å.

Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

International Nuclear Information System (INIS)

Rosenthal, Cindy; Mueller, Uwe; Panjikar, Santosh; Sun, Lianli; Ruppert, Martin; Zhao, Yu; Stöckigt, Joachim

2006-01-01

Perakine reductase, a novel member of the aldo-keto reductase enzyme superfamily of higher plants, is involved in the biosynthesis of monoterpenoid indole alkaloids in the Indian medicinal plant Rauvolfia serpentina. The enzyme has been crystallized in C-centered orthorhombic space group and diffracts to 2.0 Å resolution. Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C222 1 and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å

Interactive surface in the PapD chaperone cleft is conserved in pilus chaperone superfamily and essential in subunit recognition and assembly.

OpenAIRE

Slonim, L N; Pinkner, J S; Brändén, C I; Hultgren, S J

1992-01-01

The assembly of adhesive pili in Gram-negative bacteria is modulated by specialized periplasmic chaperone systems. PapD is the prototype member of the superfamily of periplasmic pilus chaperones. Previously, the alignment of chaperone sequences superimposed on the three dimensional structure of PapD revealed the presence of invariant, conserved and variable amino acids. Representative residues that protruded into the PapD cleft were targeted for site directed mutagenesis to investigate the pi...

Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein

International Nuclear Information System (INIS)

Klinglmayr, Eva; Wenger, Julia; Mayr, Sandra; Bossy-Wetzel, Ella; Puehringer, Sandra

2012-01-01

The crystallization and initial diffraction analysis of human Drp1 GTPase-GED fusion protein are reported. The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 Å resolution. The hexagonal-shaped crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 Å, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis

Purification and functional characterization of a protein: Bombyx mori human growth hormone like protein in silkworm pupa.

Directory of Open Access Journals (Sweden)

Jianqing Chen

Full Text Available Human growth hormone (hGH is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the β-trefoil superfamily, with β domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people's interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs.

Recombinant expression and purification of the RNA-binding LARP6 proteins from fish genetic model organisms.

Science.gov (United States)

Castro, José M; Horn, Daniel A; Pu, Xinzhu; Lewis, Karen A

2017-06-01

The RNA-binding proteins that comprise the La-related protein (LARP) superfamily have been implicated in a wide range of cellular functions, from tRNA maturation to regulation of protein synthesis. To more expansively characterize the biological function of the LARP6 subfamily, we have recombinantly expressed the full-length LARP6 proteins from two teleost fish, platyfish (Xiphophorus maculatus) and zebrafish (Danio rerio). The yields of the recombinant proteins were enhanced to >2 mg/L using a tandem approach of an N-terminal His 6 -SUMO tag and an iterative solubility screening assay to identify structurally stabilizing buffer components. The domain topologies of the purified fish proteins were probed with limited proteolysis. The fish proteins contain an internal, protease-resistant 40 kDa domain, which is considerably more stable than the comparable domain from the human LARP6 protein. The fish proteins are therefore a lucrative model system in which to study both the evolutionary divergence of this family of La-related proteins and the structure and conformational dynamics of the domains that comprise the LARP6 protein. Copyright © 2017 Elsevier Inc. All rights reserved.

HRR25, a putative protein kinase from budding yeast: Association with repair of damaged DNA

International Nuclear Information System (INIS)

Hoekstra, M.F.; Ou, A.C.; DeMaggio, A.J.; Burbee, D.G.; Liskay, R.M.; Heffron, F.

1991-01-01

In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH 2 -terminus of HRR25 contains the hallmark features of protein kinases, whereas the COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily

RTA, a candidate G protein-coupled receptor: Cloning, sequencing, and tissue distribution

International Nuclear Information System (INIS)

Ross, P.C.; Figler, R.A.; Corjay, M.H.; Barber, C.M.; Adam, N.; Harcus, D.R.; Lynch, K.R.

1990-01-01

Genomic and cDNA clones, encoding a protein that is a member of the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor superfamily, were isolated by screening rat genomic and thoracic aorta cDNA libraries with an oligonucleotide encoding a highly conserved region of the M 1 muscarinic acetylcholine receptor. Sequence analyses of these clones showed that they encode a 343-amino acid protein (named RTA). The RTA gene is single copy, as demonstrated by restriction mapping and Southern blotting of genomic clones and rat genomic DNA. RTA RNA sequences are relatively abundant throughout the gut, vas deferens, uterus, and aorta but are only barely detectable (on Northern blots) in liver, kidney, lung, and salivary gland. In the rat brain, RTA sequences are markedly abundant in the cerebellum. TRA is most closely related to the mas oncogene (34% identity), which has been suggested to be a forebrain angiotensin receptor. They conclude that RTA is not an angiotensin receptor; to date, they have been unable to identify its ligand

Vectorial loading of processive motor proteins: implementing a landscape picture

International Nuclear Information System (INIS)

Kim, Young C; Fisher, Michael E

2005-01-01

Individual processive molecular motors, of which conventional kinesin is the most studied quantitatively, move along polar molecular tracks and, by exerting a force F = (F x ,F y ,F z ) on a tether, drag cellular cargoes, in vivo, or spherical beads, in vitro, taking up to hundreds of nanometre-scale steps. From observations of velocities and the dispersion of displacements with time, under measured forces and controlled fuel supply (typically ATP), one may hope to obtain insight into the molecular motions undergone in the individual steps. In the simplest situation, the load force F may be regarded as a scalar resisting force, F x z >0, while more recently Block and co-workers (2002 Biophys. J. 83 491, 2003 Proc. Natl Acad. Sci. USA 100 2351) and Carter and Cross (2005 Nature 435 308) have studied assisting (or reverse) loads, F x >0, and also sideways (or transverse) loads F y ≠ 0. We extend previous mechanochemical kinetic models by explicitly implementing a free-energy landscape picture in order to allow for the full vectorial nature of the force F transmitted by the tether. The load-dependence of the various forward and reverse transition rates is embodied in load distribution vectors, θ j + and θ j - , which relate to substeps of the motor, and in next order, in compliance matrices η j + and η j - . The approach is applied specifically to discuss the experiments of Howard and co-workers (1996 Biophys.?J. 70 418) in which the buckling of partially clamped microtubules was measured under the action of bound kinesin molecules which induced determined perpendicular loads. But in the normal single-bead assay it also proves imperative to allow for F z >0: the appropriate analysis for kinesin, suggesting that the motor 'crouches' on binding ATP prior to stepping, is sketched. It yields an expression for the velocity, V (F x ,F z ;[ATP]), needed to address the buckling experiments

Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level.

Science.gov (United States)

Fischer, Jana; Kleinau, Gunnar; Rutz, Claudia; Zwanziger, Denise; Khajavi, Noushafarin; Müller, Anne; Rehders, Maren; Brix, Klaudia; Worth, Catherine L; Führer, Dagmar; Krude, Heiko; Wiesner, Burkhard; Schülein, Ralf; Biebermann, Heike

2018-06-01

G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of G q/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

NCBI nr-aa BLAST: CBRC-TTRU-01-0351 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-TTRU-01-0351 ref|YP_001278109.1| histone deacetylase superfamily protein [Rose...iflexus sp. RS-1] gb|ABQ92159.1| histone deacetylase superfamily [Roseiflexus sp. RS-1] YP_001278109.1 5.4 28% ...

Myostatin inhibits eEF2K-eEF2 by regulating AMPK to suppress protein synthesis.

Science.gov (United States)

Deng, Zhao; Luo, Pei; Lai, Wen; Song, Tongxing; Peng, Jian; Wei, Hong-Kui

2017-12-09

Growth of skeletal muscle is dependent on the protein synthesis, and the rate of protein synthesis is mainly regulated in the stage of translation initiation and elongation. Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, is a negative regulator of protein synthesis. C2C12 myotubes was incubated with 0, 0.01, 0.1, 1, 2, 3 μg/mL myostatin recombinant protein, and then we detected the rates of protein synthesis by the method of SUnSET. We found that high concentrations of myostatin (2 and 3 μg/mL) inhibited protein synthesis by blocking mTOR and eEF2K-eEF2 pathway, while low concentration of myostatin (0.01, 0.1 and 1 μg/mL) regulated eEF2K-eEF2 pathway activity to block protein synthesis without affected mTOR pathway, and myostatin inhibited eEF2K-eEF2 pathway through regulating AMPK pathway to suppress protein synthesis. It provided a new mechanism for myostatin regulating protein synthesis and treating muscle atrophy. Copyright © 2017. Published by Elsevier Inc.

Protein Interaction Analysis Provides a Map of the Spatial and Temporal Organization of the Ciliary Gating Zone.

Science.gov (United States)

Takao, Daisuke; Wang, Liang; Boss, Allison; Verhey, Kristen J

2017-08-07

The motility and signaling functions of the primary cilium require a unique protein and lipid composition that is determined by gating mechanisms localized at the base of the cilium. Several protein complexes localize to the gating zone and may regulate ciliary protein composition; however, the mechanisms of ciliary gating and the dynamics of the gating components are largely unknown. Here, we used the BiFC (bimolecular fluorescence complementation) assay and report for the first time on the protein-protein interactions that occur between ciliary gating components and transiting cargoes during ciliary entry. We find that the nucleoporin Nup62 and the C termini of the nephronophthisis (NPHP) proteins NPHP4 and NPHP5 interact with the axoneme-associated kinesin-2 motor KIF17 and thus spatially map to the inner region of the ciliary gating zone. Nup62 and NPHP4 exhibit rapid turnover at the transition zone and thus define dynamic components of the gate. We find that B9D1, AHI1, and the N termini of NPHP4 and NPHP5 interact with the transmembrane protein SSTR3 and thus spatially map to the outer region of the ciliary gating zone. B9D1, AHI1, and NPHP5 exhibit little to no turnover at the transition zone and thus define components of a stable gating structure. These data provide the first comprehensive map of the molecular orientations of gating zone components along the inner-to-outer axis of the ciliary gating zone. These results advance our understanding of the functional roles of gating zone components in regulating ciliary protein composition. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Role of Immunoglobulin Superfamily Cell Adhesion Molecules in Cancer Metastasis

Directory of Open Access Journals (Sweden)

Chee Wai Wong

2012-01-01

Full Text Available Metastasis is a major clinical problem and results in a poor prognosis for most cancers. The metastatic pathway describes the process by which cancer cells give rise to a metastatic lesion in a new tissue or organ. It consists of interconnecting steps all of which must be successfully completed to result in a metastasis. Cell-cell adhesion is a key aspect of many of these steps. Adhesion molecules belonging to the immunoglobulin superfamily (Ig-SF commonly play a central role in cell-cell adhesion, and a number of these molecules have been associated with cancer progression and a metastatic phenotype. Surprisingly, the contribution of Ig-SF members to metastasis has not received the attention afforded other cell adhesion molecules (CAMs such as the integrins. Here we examine the steps in the metastatic pathway focusing on how the Ig-SF members, melanoma cell adhesion molecule (MCAM, L1CAM, neural CAM (NCAM, leukocyte CAM (ALCAM, intercellular CAM-1 (ICAM-1 and platelet endothelial CAM-1 (PECAM-1 could play a role. Although much remains to be understood, this review aims to raise the profile of Ig-SF members in metastasis formation and prompt further research that could lead to useful clinical outcomes.

Identification of cytoskeleton-associated proteins essential for lysosomal stability and survival of human cancer cells

DEFF Research Database (Denmark)

Groth-Pedersen, Line; Aits, Sonja; Corcelle-Termeau, Elisabeth

2012-01-01

Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library...... in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 si......), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, i.e. photo-oxidation, siramesine, etoposide...

Full-Length Venom Protein cDNA Sequences from Venom-Derived mRNA: Exploring Compositional Variation and Adaptive Multigene Evolution.

Science.gov (United States)

Modahl, Cassandra M; Mackessy, Stephen P

2016-06-01

Envenomation of humans by snakes is a complex and continuously evolving medical emergency, and treatment is made that much more difficult by the diverse biochemical composition of many venoms. Venomous snakes and their venoms also provide models for the study of molecular evolutionary processes leading to adaptation and genotype-phenotype relationships. To compare venom complexity and protein sequences, venom gland transcriptomes are assembled, which usually requires the sacrifice of snakes for tissue. However, toxin transcripts are also present in venoms, offering the possibility of obtaining cDNA sequences directly from venom. This study provides evidence that unknown full-length venom protein transcripts can be obtained from the venoms of multiple species from all major venomous snake families. These unknown venom protein cDNAs are obtained by the use of primers designed from conserved signal peptide sequences within each venom protein superfamily. This technique was used to assemble a partial venom gland transcriptome for the Middle American Rattlesnake (Crotalus simus tzabcan) by amplifying sequences for phospholipases A2, serine proteases, C-lectins, and metalloproteinases from within venom. Phospholipase A2 sequences were also recovered from the venoms of several rattlesnakes and an elapid snake (Pseudechis porphyriacus), and three-finger toxin sequences were recovered from multiple rear-fanged snake species, demonstrating that the three major clades of advanced snakes (Elapidae, Viperidae, Colubridae) have stable mRNA present in their venoms. These cDNA sequences from venom were then used to explore potential activities derived from protein sequence similarities and evolutionary histories within these large multigene superfamilies. Venom-derived sequences can also be used to aid in characterizing venoms that lack proteomic profiles and identify sequence characteristics indicating specific envenomation profiles. This approach, requiring only venom, provides

Data growth and its impact on the SCOP database: new developments

Energy Technology Data Exchange (ETDEWEB)

Chandonia, John-Marc; Andreeva, Antonina; Howorth, Dave; Chandonia, John-Marc; Brenner, Steven E.; Hubbard, Tim J.P.; Chothia, Cyrus; Murzin, Alexey G.

2007-11-13

The Structural Classification of Proteins (SCOP) database is a comprehensive ordering of all proteins of known structure, according to their evolutionary and structural relationships. The SCOP hierarchy comprises the following levels: Species, Protein, Family, Superfamily, Fold and Class. While keeping the original classification scheme intact, we have changed the production of SCOP in order to cope with a rapid growth of new structural data and to facilitate the discovery of new protein relationships. We describe ongoing developments and new features implemented in SCOP. A new update protocol supports batch classification of new protein structuresby their detected relationships at Family and Superfamily levels in contrast to our previous sequential handling of new structural data by release date. We introduce pre-SCOP, a preview of the SCOP developmental version that enables earlier access to the information on new relationships. We also discuss the impact of worldwide Structural Genomics initiatives, which are producing new protein structures at an increasing rate, on the rates of discovery and growth of protein families and superfamilies. SCOP can be accessed at http://scop.mrc-lmb.cam.ac.uk/scop.

The Drosophila KIF1A homolog unc-104 is important for site-specific active zone maturation

Directory of Open Access Journals (Sweden)

Yao V. Zhang

2016-09-01

Full Text Available Abstract Mutations in the kinesin-3 family member KIF1A have been associated with hereditary spastic paraplegia, hereditary sensory and autonomic neuropathy type 2 and intellectual disability. Both autosomal recessive and autosomal dominant forms of inheritance have been reported. Loss of KIF1A or its homolog unc-104 causes early postnatal or embryonic lethality in mice and Drosophila, respectively. In this study we use a previously described hypomorphic allele of unc-104, unc-104bris, to investigate the impact of partial loss-of-function of kinesin-3 function on active zone formation at the Drosophila neuromuscular junction. unc-104bris mutants exhibit synaptic defects where a subset of synapses at the neuromuscular junction lack the key active zone organizer protein Bruchpilot. Modulating synaptic Bruchpilot levels by ectopic overexpression or RNAi-mediated knockdown suggests that the loss of active zone components such as Ca2+ channel and Liprin-α from these synapses is caused by impaired kinesin-3 transport rather than due to the absence of Bruchpilot at these synapses. In addition to defects in active zone maturation, unc-104bris mutants display impaired transport of dense core vesicles and synaptic vesicle associated proteins, among which Rab3 has been shown to regulate the distribution of Bruchpilot to active zones. Overexpression of Rab3 partially ameliorates synaptic phenotypes of unc-104bris neuromuscular junction, suggesting that lack of presynaptic Rab3 may contribute to defects in synapse maturation.

Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella Typhi

DEFF Research Database (Denmark)

Shaheen, Aqsa; Ismat, Fouzia; Iqbal, Mazhar

2015-01-01

Multidrug resistance mediated by efflux pumps is a well-known phenomenon in infectious bacteria. Although much work has been carried out to characterize multidrug efflux pumps in Gram-negative and Gram-positive bacteria, such information is still lacking for many deadly pathogens. The aim...... of this study was to gain insight into the substrate specificity of previously uncharacterized transporters of Salmonella Typhi to identify their role in the development of multidrug resistance. S. Typhi genes encoding putative members of the major facilitator superfamily were cloned and expressed in the drug......-hypersensitive Escherichia coli strain KAM42, and tested for transport of 25 antibacterial compounds, including representative antibiotics of various classes, antiseptics, dyes and detergents. Of the 15 tested putative transporters, STY0901, STY2458 and STY4874 exhibited a drug-resistance phenotype. Among these, STY4874...

Integrated Approaches for Genome-wide Interrogation of the Druggable Non-olfactory G Protein-coupled Receptor Superfamily.

Science.gov (United States)

Roth, Bryan L; Kroeze, Wesley K

2015-08-07

G-protein-coupled receptors (GPCRs) are frequent and fruitful targets for drug discovery and development, as well as being off-targets for the side effects of a variety of medications. Much of the druggable non-olfactory human GPCR-ome remains under-interrogated, and we present here various approaches that we and others have used to shine light into these previously dark corners of the human genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Impact of G protein-coupled receptor heteromers in endocrine systems.

Science.gov (United States)

Jonas, K C; Hanyaloglu, A C

2017-07-05

The fine-tuning of endocrine homeostasis is regulated by dynamic receptor mediated processes. The superfamily of G protein-coupled receptors (GPCRs) have diverse roles in the modulation of all endocrine axes, thus understanding the mechanisms underpinning their functionality is paramount for treatment of endocrinopathies. Evidence over the last 20 years has highlighted homo and heteromerization as a key mode of mediating GPCR functional diversity. This review will discuss the concept of GPCR heteromerization and its relevance to endocrine function, detailing in vitro and in vivo evidence, and exploring current and potential pharmacological strategies for specific targeting of GPCR heteromers in endocrine heath and disease. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Dynamic expression pattern of kinesin accessory protein in Drosophila

Indian Academy of Sciences (India)

Unknown

terization of the function of the DmKAP gene, we studied its expression pattern at different stages of development using the mRNA in .... region of the developing brain. ..... Kido M and Hirokawa N 1998 Randomization of left-right asymmetry ...

Evolution of function in the "two dinucleotide binding domains" flavoproteins.

Directory of Open Access Journals (Sweden)

Sunil Ojha

2007-07-01

Full Text Available Structural and biochemical constraints force some segments of proteins to evolve more slowly than others, often allowing identification of conserved structural or sequence motifs that can be associated with substrate binding properties, chemical mechanisms, and molecular functions. We have assessed the functional and structural constraints imposed by cofactors on the evolution of new functions in a superfamily of flavoproteins characterized by two-dinucleotide binding domains, the "two dinucleotide binding domains" flavoproteins (tDBDF superfamily. Although these enzymes catalyze many different types of oxidation/reduction reactions, each is initiated by a stereospecific hydride transfer reaction between two cofactors, a pyridine nucleotide and flavin adenine dinucleotide (FAD. Sequence and structural analysis of more than 1,600 members of the superfamily reveals new members and identifies details of the evolutionary connections among them. Our analysis shows that in all of the highly divergent families within the superfamily, these cofactors adopt a conserved configuration optimal for stereospecific hydride transfer that is stabilized by specific interactions with amino acids from several motifs distributed among both dinucleotide binding domains. The conservation of cofactor configuration in the active site restricts the pyridine nucleotide to interact with FAD from the re-side, limiting the flow of electrons from the re-side to the si-side. This directionality of electron flow constrains interactions with the different partner proteins of different families to occur on the same face of the cofactor binding domains. As a result, superimposing the structures of tDBDFs aligns not only these interacting proteins, but also their constituent electron acceptors, including heme and iron-sulfur clusters. Thus, not only are specific aspects of the cofactor-directed chemical mechanism conserved across the superfamily, the constraints they impose are

The G protein-coupled receptor subset of the dog genome is more similar to that in humans than rodents

OpenAIRE

Schiöth Helgi B; Foord Steven M; Fredriksson Robert; Haitina Tatjana; Gloriam David E

2009-01-01

Abstract Background The dog is an important model organism and it is considered to be closer to humans than rodents regarding metabolism and responses to drugs. The close relationship between humans and dogs over many centuries has lead to the diversity of the canine species, important genetic discoveries and an appreciation of the effects of old age in another species. The superfamily of G protein-coupled receptors (GPCRs) is one of the largest gene families in most mammals and the most expl...

Plant virus cell-to-cell movement is not dependent on the transmembrane disposition of its movement protein.

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Martínez-Gil, Luis; Sánchez-Navarro, Jesús A; Cruz, Antonio; Pallás, Vicente; Pérez-Gil, Jesús; Mingarro, Ismael

2009-06-01

The cell-to-cell transport of plant viruses depends on one or more virus-encoded movement proteins (MPs). Some MPs are integral membrane proteins that interact with the membrane of the endoplasmic reticulum, but a detailed understanding of the interaction between MPs and biological membranes has been lacking. The cell-to-cell movement of the Prunus necrotic ringspot virus (PNRSV) is facilitated by a single MP of the 30K superfamily. Here, using a myriad of biochemical and biophysical approaches, we show that the PNRSV MP contains only one hydrophobic region (HR) that interacts with the membrane interface, as opposed to being a transmembrane protein. We also show that a proline residue located in the middle of the HR constrains the structural conformation of this region at the membrane interface, and its replacement precludes virus movement.

The genome sequence of the protostome Daphnia pulex encodes respective orthologues of a neurotrophin, a Trk and a p75NTR: Evolution of neurotrophin signaling components and related proteins in the bilateria

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Wilson Karen HS

2009-10-01

Full Text Available Abstract Background Neurotrophins and their Trk and p75NTR receptors play an important role in the nervous system. To date, neurotrophins, Trk and p75NTR have only been found concomitantly in deuterostomes. In protostomes, homologues to either neurotrophin, Trk or p75NTR are reported but their phylogenetic relationship to deuterostome neurotrophin signaling components is unclear. Drosophila has neurotrophin homologues called Spätzles (Spz, some of which were recently renamed neurotrophins, but direct proof that these are deuterostome neurotrophin orthologues is lacking. Trks belong to the receptor tyrosine kinase (RTK family and among RTKs, Trks and RORs are closest related. Flies lack Trks but have ROR and ROR-related proteins called NRKs playing a neurotrophic role. Mollusks have so far the most similar proteins to Trks (Lymnaea Trk and Aplysia Trkl but the exact phylogenetic relationship of mollusk Trks to each other and to vertebrate Trks is unknown. p75NTR belongs to the tumor necrosis factor receptor (TNFR superfamily. The divergence of the TNFR families in vertebrates has been suggested to parallel the emergence of the adaptive immune system. Only one TNFR representative, the Drosophila Wengen, has been found in protostomes. To clarify the evolution of neurotrophin signaling components in bilateria, this work analyzes the genome of the crustacean Daphnia pulex as well as new genetic data from protostomes. Results The Daphnia genome encodes a neurotrophin, p75NTR and Trk orthologue together with Trkl, ROR, and NRK-RTKs. Drosophila Spz1, 2, 3, 5, 6 orthologues as well as two new groups of Spz proteins (Spz7 and 8 are also found in the Daphnia genome. Searching genbank and the genomes of Capitella, Helobdella and Lottia reveals neurotrophin signaling components in other protostomes. Conclusion It appears that a neurotrophin, Trk and p75NTR existed at the protostome/deuterostome split. In protostomes, a "neurotrophin superfamily" includes

Phi Class of Glutathione S-transferase Gene Superfamily Widely Exists in Nonplant Taxonomic Groups.

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Munyampundu, Jean-Pierre; Xu, You-Ping; Cai, Xin-Zhong

2016-01-01

Glutathione S-transferases (GSTs) constitute a superfamily of enzymes involved in detoxification of noxious compounds and protection against oxidative damage. GST class Phi (GSTF), one of the important classes of plant GSTs, has long been considered as plant specific but was recently found in basidiomycete fungi. However, the range of nonplant taxonomic groups containing GSTFs remains unknown. In this study, the distribution and phylogenetic relationships of nonplant GSTFs were investigated. We identified GSTFs in ascomycete fungi, myxobacteria, and protists Naegleria gruberi and Aureococcus anophagefferens. GSTF occurrence in these bacteria and protists correlated with their genome sizes and habitats. While this link was missing across ascomycetes, the distribution and abundance of GSTFs among ascomycete genomes could be associated with their lifestyles to some extent. Sequence comparison, gene structure, and phylogenetic analyses indicated divergence among nonplant GSTFs, suggesting polyphyletic origins during evolution. Furthermore, in silico prediction of functional partners suggested functional diversification among nonplant GSTFs.

Interplay between microtubule bundling and sorting factors ensures acentriolar spindle stability during C. elegans oocyte meiosis.

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Timothy J Mullen

2017-09-01

Full Text Available In many species, oocyte meiosis is carried out in the absence of centrioles. As a result, microtubule organization, spindle assembly, and chromosome segregation proceed by unique mechanisms. Here, we report insights into the principles underlying this specialized form of cell division, through studies of C. elegans KLP-15 and KLP-16, two highly homologous members of the kinesin-14 family of minus-end-directed kinesins. These proteins localize to the acentriolar oocyte spindle and promote microtubule bundling during spindle assembly; following KLP-15/16 depletion, microtubule bundles form but then collapse into a disorganized array. Surprisingly, despite this defect we found that during anaphase, microtubules are able to reorganize into a bundled array that facilitates chromosome segregation. This phenotype therefore enabled us to identify factors promoting microtubule organization during anaphase, whose contributions are normally undetectable in wild-type worms; we found that SPD-1 (PRC1 bundles microtubules and KLP-18 (kinesin-12 likely sorts those bundles into a functional orientation capable of mediating chromosome segregation. Therefore, our studies have revealed an interplay between distinct mechanisms that together promote spindle formation and chromosome segregation in the absence of structural cues such as centrioles.

Automatic structure classification of small proteins using random forest

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Hirst Jonathan D

2010-07-01

Full Text Available Abstract Background Random forest, an ensemble based supervised machine learning algorithm, is used to predict the SCOP structural classification for a target structure, based on the similarity of its structural descriptors to those of a template structure with an equal number of secondary structure elements (SSEs. An initial assessment of random forest is carried out for domains consisting of three SSEs. The usability of random forest in classifying larger domains is demonstrated by applying it to domains consisting of four, five and six SSEs. Results Random forest, trained on SCOP version 1.69, achieves a predictive accuracy of up to 94% on an independent and non-overlapping test set derived from SCOP version 1.73. For classification to the SCOP Class, Fold, Super-family or Family levels, the predictive quality of the model in terms of Matthew's correlation coefficient (MCC ranged from 0.61 to 0.83. As the number of constituent SSEs increases the MCC for classification to different structural levels decreases. Conclusions The utility of random forest in classifying domains from the place-holder classes of SCOP to the true Class, Fold, Super-family or Family levels is demonstrated. Issues such as introduction of a new structural level in SCOP and the merger of singleton levels can also be addressed using random forest. A real-world scenario is mimicked by predicting the classification for those protein structures from the PDB, which are yet to be assigned to the SCOP classification hierarchy.

The function of yeast CAP family proteins in lipid export, mating, and pathogen defense.

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Darwiche, Rabih; El Atab, Ola; Cottier, Stéphanie; Schneiter, Roger

2018-04-01

In their natural habitat, yeast cells are constantly challenged by changing environmental conditions and a fierce competition for limiting resources. To thrive under such conditions, cells need to adapt and divide quickly, and be able to neutralize the toxic compounds secreted by their neighbors. Proteins like the pathogen-related yeast, Pry proteins, which belong to the large CAP/SCP/TAPS superfamily, may have an important role in this function. CAP proteins are conserved from yeast to man and are characterized by a unique αβα sandwich fold. They are mostly secreted glycoproteins and have been implicated in many different physiological processes including pathogen defense, virulence, venom toxicity, and sperm maturation. Yeast members of this family bind and export sterols as well as fatty acids, and they render cells resistant to eugenol, an antimicrobial compound present in clove oil. CAP family members might thus exert their various physiological functions through binding, sequestration, and neutralization of such small hydrophobic compounds. © 2017 Federation of European Biochemical Societies.

Novel evolutionary lineages of the invertebrate oxytocin/vasopressin superfamily peptides and their receptors in the common octopus (Octopus vulgaris)

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Kanda, Atsuhiro; Satake, Honoo; Kawada, Tsuyoshi; Minakata, Hiroyuki

2004-01-01

The common octopus, Octopus vulgaris, is the first invertebrate species that was shown to possess two oxytocin/vasopressin (OT/VP) superfamily peptides, octopressin (OP) and cephalotocin (CT). Previously, we cloned a GPCR (G-protein-coupled receptor) specific to CT [CTR1 (CT receptor 1)]. In the present study, we have identified an additional CTR, CTR2, and a novel OP receptor, OPR. Both CTR2 and OPR include domains and motifs typical of GPCRs, and the intron– exon structures are in accord with those of OT/VP receptor genes. CTR2 and OPR expressed in Xenopus oocytes induced calcium-mediated inward chloride current in a CT- and OP-specific manner respectively. Several regions and residues, which are requisite for binding of the vertebrate OT/VP receptor family with their ligands, are highly conserved in CTRs, but not in OPR. These different sequences between CTRs and OPR, as well as the amino acid residues of OP and CT at positions 2–5, were presumed to play crucial roles in the binding selectivity to their receptors, whereas the difference in the polarity of OT/VP family peptide residues at position 8 confers OT and VP with the binding specificity in vertebrates. CTR2 mRNA was present in various peripheral tissues, and OPR mRNA was detected in both the nervous system and peripheral tissues. Our findings suggest that the CT and OP genes, similar to the OT/VP family, evolved through duplication, but the ligand–receptor selectivity were established through different evolutionary lineages from those of their vertebrate counterparts. PMID:15504101

Secretoglobin Superfamily Protein SCGB3A2 Deficiency Potentiates Ovalbumin-Induced Allergic Pulmonary Inflammation

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Taketomo Kido

2014-01-01

Full Text Available Secretoglobin (SCGB 3A2, a cytokine-like secretory protein of small molecular weight, which may play a role in lung inflammation, is predominantly expressed in airway epithelial cells. In order to understand the physiological role of SCGB3A2, Scgb3a2−/− mice were generated and characterized. Scgb3a2−/− mice did not exhibit any overt phenotypes. In ovalbumin- (OVA- induced airway allergy inflammation model, Scgb3a2−/− mice in mixed background showed a decreased OVA-induced airway inflammation, while six times C57BL/6NCr backcrossed congenic Scgb3a2−/− mice showed a slight exacerbation of OVA-induced airway inflammation as compared to wild-type littermates. These results indicate that the loss of SCGB3A2 function was influenced by a modifier gene(s in mixed genetic background and suggest that SCGB3A2 has anti-inflammatory property. The results further suggest the possible use of recombinant human SCGB3A2 as an anti-inflammatory agent.

Kernel based machine learning algorithm for the efficient prediction of type III polyketide synthase family of proteins

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Mallika V

2010-03-01

Full Text Available Type III Polyketide synthases (PKS are family of proteins considered to have significant role in the biosynthesis of various polyketides in plants, fungi and bacteria. As these proteins show positive effects to human health, more researches are going on regarding this particular protein. Developing a tool to identify the probability of sequence, being a type III polyketide synthase will minimize the time consumption and manpower efforts. In this approach, we have designed and implemented PKSIIIpred, a high performance prediction server for type III PKS where the classifier is Support Vector Machine (SVM. Based on the limited training dataset, the tool efficiently predicts the type III PKS superfamily of proteins with high sensitivity and specificity. PKSIIIpred is available at http://type3pks.in/prediction/. We expect that this tool may serve as a useful resource for type III PKS researchers. Currently work is being progressed for further betterment of prediction accuracy by including more sequence features in the training dataset.

Characterization of pathogenic germline mutations in human Protein Kinases

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Orengo Christine A

2011-07-01

Full Text Available Abstract Background Protein Kinases are a superfamily of proteins involved in crucial cellular processes such as cell cycle regulation and signal transduction. Accordingly, they play an important role in cancer biology. To contribute to the study of the relation between kinases and disease we compared pathogenic mutations to neutral mutations as an extension to our previous analysis of cancer somatic mutations. First, we analyzed native and mutant proteins in terms of amino acid composition. Secondly, mutations were characterized according to their potential structural effects and finally, we assessed the location of the different classes of polymorphisms with respect to kinase-relevant positions in terms of subfamily specificity, conservation, accessibility and functional sites. Results Pathogenic Protein Kinase mutations perturb essential aspects of protein function, including disruption of substrate binding and/or effector recognition at family-specific positions. Interestingly these mutations in Protein Kinases display a tendency to avoid structurally relevant positions, what represents a significant difference with respect to the average distribution of pathogenic mutations in other protein families. Conclusions Disease-associated mutations display sound differences with respect to neutral mutations: several amino acids are specific of each mutation type, different structural properties characterize each class and the distribution of pathogenic mutations within the consensus structure of the Protein Kinase domain is substantially different to that for non-pathogenic mutations. This preferential distribution confirms previous observations about the functional and structural distribution of the controversial cancer driver and passenger somatic mutations and their use as a proxy for the study of the involvement of somatic mutations in cancer development.

Shortening actin filaments cause force generation in actomyosin network to change from contractile to extensile

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Kumar, Nitin; Gardel, Margaret

Motor proteins in conjunction with filamentous proteins convert biochemical energy into mechanical energy which serves a number of cellular processes including cell motility, force generation and intracellular cargo transport. In-vitro experiments suggest that the forces generated by kinesin motors on microtubule bundles are extensile in nature whereas myosin motors on actin filaments are contractile. It is not clear how qualitatively similar systems can show completely different behaviors in terms of the nature of force generation. In order to answer this question, we carry out in vitro experiments where we form quasi 2D filamentous actomyosin networks and vary the length of actin filaments by adding capping protein. We show that when filaments are much shorter than their typical persistence length (approximately 10 microns), the forces generated are extensile and we see active nematic defect propagation, as seen in the microtubule-kinesin system. Based on this observation, we claim that the rigidity of rods plays an important role in dictating the nature of force generation in such systems. In order to understand this transition, we selectively label individual filaments and find that longer filaments show considerable bending and buckling, making them difficult to slide and extend along their length.

Electrophoretic mobility shift in native gels indicates calcium-dependent structural changes of neuronal calcium sensor proteins.

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Viviano, Jeffrey; Krishnan, Anuradha; Wu, Hao; Venkataraman, Venkat

2016-02-01

In proteins of the neuronal calcium sensor (NCS) family, changes in structure as well as function are brought about by the binding of calcium. In this article, we demonstrate that these structural changes, solely due to calcium binding, can be assessed through electrophoresis in native gels. The results demonstrate that the NCS proteins undergo ligand-dependent conformational changes that are detectable in native gels as a gradual decrease in mobility with increasing calcium but not other tested divalent cations such as magnesium, strontium, and barium. Surprisingly, such a gradual change over the entire tested range is exhibited only by the NCS proteins but not by other tested calcium-binding proteins such as calmodulin and S100B, indicating that the change in mobility may be linked to a unique NCS family feature--the calcium-myristoyl switch. Even within the NCS family, the changes in mobility are characteristic of the protein, indicating that the technique is sensitive to the individual features of the protein. Thus, electrophoretic mobility on native gels provides a simple and elegant method to investigate calcium (small ligand)-induced structural changes at least in the superfamily of NCS proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Caractérisation biochimique et fonctionnelle de glutathion-S-transferases (GSTs) chez Phanerochaete chrysosporium

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Anak Ngadin , Andrew

2011-01-01

Phanerochaete chrysosporium is a ligninolytic fungus widely studied because of its capacities to degrade wood and xenobiotics through an extracellular enzymatic system. Its genome has been sequenced and has provided researchers with a complete inventory of the predicted proteins produced by this organism. This has allowed the description of many protein superfamilies. Among them, Glutathione S-transferases (GSTs) constitute a complex and widespread superfamily classified as enzymes of seconda...

Genetic polymorphisms in glutathione S-transferase (GST) superfamily and arsenic metabolism in residents of the Red River Delta, Vietnam

International Nuclear Information System (INIS)

Agusa, Tetsuro; Iwata, Hisato; Fujihara, Junko; Kunito, Takashi; Takeshita, Haruo; Tu Binh Minh; Pham Thi Kim Trang; Pham Hung Viet; Tanabe, Shinsuke

2010-01-01

To elucidate the role of genetic factors in arsenic metabolism, we investigated associations of genetic polymorphisms in the members of glutathione S-transferase (GST) superfamily with the arsenic concentrations in hair and urine, and urinary arsenic profile in residents in the Red River Delta, Vietnam. Genotyping was conducted for GST ω1 (GSTO1) Ala140Asp, Glu155del, Glu208Lys, Thr217Asn, and Ala236Val, GST ω2 (GSTO2) Asn142Asp, GST π1 (GSTP1) Ile105Val, GST μ1 (GSTM1) wild/null, and GST θ1 (GSTT1) wild/null. There were no mutation alleles for GSTO1 Glu208Lys, Thr217Asn, and Ala236Val in this population. GSTO1 Glu155del hetero type showed higher urinary concentration of As V than the wild homo type. Higher percentage of DMA V in urine of GSTM1 wild type was observed compared with that of the null type. Strong correlations between GSTP1 Ile105Val and arsenic exposure level and profile were observed in this study. Especially, heterozygote of GSTP1 Ile105Val had a higher metabolic capacity from inorganic arsenic to monomethyl arsenic, while the opposite trend was observed for ability of metabolism from As V to As III . Furthermore, other factors including sex, age, body mass index, arsenic level in drinking water, and genotypes of As (+ 3 oxidation state) methyltransferase (AS3MT) were also significantly co-associated with arsenic level and profile in the Vietnamese. To our knowledge, this is the first study indicating the associations of genetic factors of GST superfamily with arsenic metabolism in a Vietnamese population.

The kinesin–tubulin complex: considerations in structural and functional complexity

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Olmsted ZT

2015-02-01

Full Text Available Zachary T Olmsted, Andrew G Colliver, Janet L Paluh State University of New York Polytechnic Institute, Colleges of Nanoscale Science and Engineering, College of Nanoscale Science, Nanobioscience Constellation, Albany, NY, USA Abstract: The ability of cells to respond to external cues by appropriately manipulating their internal environment requires a dynamic microtubule cytoskeleton that is facilitated by associated kinesin motor interactions. The evolutionary adaptations of kinesins and tubulins when merged generate a highly adaptable communication and infrastructure cellular network that is important to understanding specialized cell functions, human disease, and disease therapies. Here, we review the state of the field in the complex relationship of kinesin–tubulin interactions. We propose 12 mechanistic specializations of kinesins. In one category, referred to as sortability, we describe how kinesin interactions with tubulin isoforms, isotypes, or posttranslationally modified tubulins contribute to diverse cellular roles. Fourteen kinesin families have previously been described. Here, we illustrate the great depth of functional complexity that is possible in members within a single kinesin family by mechanistic specialization through discussion of the well-studied Kinesin-14 family. This includes new roles of Kinesin-14 in regulating supramolecular structures such as the microtubule-organizing center γ-tubulin ring complex of centrosomes. We next explore the value of an improved mechanistic understanding of kinesin–tubulin interactions in regard to human development, disease mechanisms, and improving treatments that target kinesin–tubulin complexes. The ability to combine the current kinesin nomenclature along with a more precisely defined kinesin and tubulin molecular toolbox is needed to support more detailed exploration of kinesin–tubulin interaction mechanisms including functional uniqueness, redundancy, or adaptations to new

Mycobacterium smegmatis SftH exemplifies a distinctive clade of superfamily II DNA-dependent ATPases with 3′ to 5′ translocase and helicase activities

OpenAIRE

Yakovleva, Lyudmila; Shuman, Stewart

2012-01-01

Bacterial DNA helicases are nucleic acid-dependent NTPases that play important roles in DNA replication, recombination and repair. We are interested in the DNA helicases of Mycobacteria, a genus of the phylum Actinobacteria, which includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis SftH, a superfamily II helicase with a distinctive domain structure, comprising an N-terminal NTPase domain and...

Aquaporin-11: A channel protein lacking apparent transport function expressed in brain

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Tsunenari Takashi

2006-05-01

Full Text Available Abstract Background The aquaporins are a family of integral membrane proteins composed of two subfamilies: the orthodox aquaporins, which transport only water, and the aquaglyceroporins, which transport glycerol, urea, or other small solutes. Two recently described aquaporins, numbers 11 and 12, appear to be more distantly related to the other mammalian aquaporins and aquaglyceroporins. Results We report on the characterization of Aquaporin-11 (AQP11. AQP11 RNA and protein is found in multiple rat tissues, including kidney, liver, testes and brain. AQP11 has a unique distribution in brain, appearing in Purkinje cell dendrites, hippocampal neurons of CA1 and CA2, and cerebral cortical neurons. Immunofluorescent staining of Purkinje cells indicates that AQP11 is intracellular. Unlike other aquaporins, Xenopus oocytes expressing AQP11 in the plasma membrane failed to transport water, glycerol, urea, or ions. Conclusion AQP11 is functionally distinct from other proteins of the aquaporin superfamily and could represent a new aquaporin subfamily. Further studies are necessary to elucidate the role of AQP11 in the brain.

Disruption of M-T5, a novel myxoma virus gene member of poxvirus host range superfamily, results in dramatic attenuation of myxomatosis in infected European rabbits.

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Mossman, K; Lee, S F; Barry, M; Boshkov, L; McFadden, G

1996-07-01

Myxoma virus is a pathogenic poxvirus that induces a lethal myxomatosis disease profile in European rabbits, which is characterized by fulminating lesions at the primary site of inoculation, rapid dissemination to secondary internal organs and peripheral external sites, and supervening gram-negative bacterial infection. Here we describe the role of a novel myxoma virus protein encoded by the M-T5 open reading frame during pathogenesis. The myxoma virus M-T5 protein possesses no significant sequence homology to nonviral proteins but is a member of a larger poxviral superfamily designated host range proteins. An M-T5- mutant virus was constructed by disruption of both copies of the M-T5 gene followed by insertion of the selectable marker p7.5Ecogpt. Although the M-T5- deletion mutant replicated with wild-type kinetics in rabbit fibroblasts, infection of a rabbit CD4+ T-cell line (RL5) with the myxoma virus M-T5- mutant virus resulted in the rapid and complete cessation of both host and viral protein synthesis, accompanied by the manifestation of all the classical features of programmed cell death. Infection of primary rabbit peripheral mononuclear cells with the myxoma virus M-T5-mutant virus resulted in the apoptotic death of nonadherent lymphocytes but not adherent monocytes. Within the European rabbit, disruption of the M-T5 open reading frame caused a dramatic attenuation of the rapidly lethal myxomatosis infection, and none of the infected rabbits displayed any of the characteristic features of myxomatosis. The two most significant histological observations in rabbits infected with the M-T5-mutant virus were (i) the lack of progression of the infection past the primary site of inoculation, coupled with the establishment of a rapid and effective inflammatory reaction, and (ii) the inability of the virus to initiate a cellular reaction within secondary immune organs. We conclude that M-T5 functions as a critical virulence factor by allowing productive infection of

Ku proteins function as corepressors to regulate farnesoid X receptor-mediated gene expression

International Nuclear Information System (INIS)

Ohno, Masae; Kunimoto, Masaaki; Nishizuka, Makoto; Osada, Shigehiro; Imagawa, Masayoshi

2009-01-01

The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST-fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR associated factors. These proteins are known to have an important role in DNA repair, recombination, and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.

Crystallization and preliminary X-ray diffraction analysis of phospholipid-bound Sfh1p, a member of the Saccharomyces cerevisiae Sec14p-like phosphatidylinositol transfer protein family

International Nuclear Information System (INIS)

Schaaf, Gabriel; Betts, Laurie; Garrett, Teresa A.; Raetz, Christian R. H.; Bankaitis, Vytas A.

2006-01-01

Yeast Sfh1p, a close homolog of the Sec14p phosphatidylinositol transfer protein, was crystallized in the absence of detergent. X-ray data have been collected to 2.5 Å. Sec14p is the major phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein in the budding yeast Saccharomyces cerevisiae and is the founding member of a large eukaryotic protein superfamily. This protein catalyzes the exchange of either PtdIns or PtdCho between membrane bilayers in vitro and this exchange reaction requires no external input of energy or of other protein cofactors. Despite the previous elucidation of the crystal structure of a detergent-bound form of Sec14p, the conformational changes that accompany the phospholipid-exchange reaction remain undefined. Moreover, a structural appreciation of how Sec14p or its homologs bind their various phospholipid substrates remains elusive. Here, the purification and crystallization of yeast Sfh1p, the protein most closely related to Sec14p, are reported. A combination of electrospray ionization mass-spectrometry and collision-induced decomposition mass-spectrometry methods indicate that recombinant Sfh1p loads predominantly with phosphatidylethanolamine. Unlike phospholipid-bound forms of Sec14p, this form of Sfh1p crystallizes readily in the absence of detergent. Sfh1p crystals diffract to 2.5 Å and belong to the orthorhombic primitive space group P2 1 2 1 2 1 , with unit-cell parameters a = 49.40, b = 71.55, c = 98.21 Å, α = β = γ = 90°. One Sfh1p molecule is present in the asymmetric unit (V M = 2.5 Å 3 Da −1 ; V s = 50%). Crystallization of a phospholipid-bound Sec14p-like protein is a critical first step in obtaining the first high-resolution picture of how proteins of the Sec14p superfamily bind their phospholipid ligands. This information will significantly extend our current understanding of how Sec14p-like proteins catalyze phospholipid exchange

Common gene variants in the tumor necrosis factor (TNF and TNF receptor superfamilies and NF-kB transcription factors and non-Hodgkin lymphoma risk.

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Sophia S Wang

Full Text Available A promoter polymorphism in the pro-inflammatory cytokine tumor necrosis factor (TNF (TNF G-308A is associated with increased non-Hodgkin lymphoma (NHL risk. The protein product, TNF-alpha, activates the nuclear factor kappa beta (NF-kappaB transcription factor, and is critical for inflammatory and apoptotic responses in cancer progression. We hypothesized that the TNF and NF-kappaB pathways are important for NHL and that gene variations across the pathways may alter NHL risk.We genotyped 500 tag single nucleotide polymorphisms (SNPs from 48 candidate gene regions (defined as 20 kb 5', 10 kb 3' in the TNF and TNF receptor superfamilies and the NF-kappaB and related transcription factors, in 1946 NHL cases and 1808 controls pooled from three independent population-based case-control studies. We obtained a gene region-level summary of association by computing the minimum p-value ("minP test". We used logistic regression to compute odds ratios and 95% confidence intervals for NHL and four major NHL subtypes in relation to SNP genotypes and haplotypes. For NHL, the tail strength statistic supported an overall relationship between the TNF/NF-kappaB pathway and NHL (p = 0.02. We confirmed the association between TNF/LTA on chromosome 6p21.3 with NHL and found the LTA rs2844484 SNP most significantly and specifically associated with the major subtype, diffuse large B-cell lymphoma (DLBCL (p-trend = 0.001. We also implicated for the first time, variants in NFKBIL1 on chromosome 6p21.3, associated with NHL. Other gene regions identified as statistically significantly associated with NHL included FAS, IRF4, TNFSF13B, TANK, TNFSF7 and TNFRSF13C. Accordingly, the single most significant SNPs associated with NHL were FAS rs4934436 (p-trend = 0.0024, IRF4 rs12211228 (p-trend = 0.0026, TNFSF13B rs2582869 (p-trend = 0.0055, TANK rs1921310 (p-trend = 0.0025, TNFSF7 rs16994592 (p-trend = 0.0024, and TNFRSF13C rs6002551 (p-trend = 0.0074. All associations were

Topology of transmembrane channel-like gene 1 protein.

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Labay, Valentina; Weichert, Rachel M; Makishima, Tomoko; Griffith, Andrew J

2010-10-05

Mutations of transmembrane channel-like gene 1 (TMC1) cause hearing loss in humans and mice. TMC1 is the founding member of a family of genes encoding proteins of unknown function that are predicted to contain multiple transmembrane domains. The goal of our study was to define the topology of mouse TMC1 expressed heterologously in tissue culture cells. TMC1 was retained in the endoplasmic reticulum (ER) membrane of five tissue culture cell lines that we tested. We used anti-TMC1 and anti-HA antibodies to probe the topologic orientation of three native epitopes and seven HA epitope tags along full-length TMC1 after selective or complete permeabilization of transfected cells with digitonin or Triton X-100, respectively. TMC1 was present within the ER as an integral membrane protein containing six transmembrane domains and cytosolic N- and C-termini. There is a large cytoplasmic loop, between the fourth and fifth transmembrane domains, with two highly conserved hydrophobic regions that might associate with or penetrate, but do not span, the plasma membrane. Our study is the first to demonstrate that TMC1 is a transmembrane protein. The topologic organization revealed by this study shares some features with that of the shaker-TRP superfamily of ion channels.

Overexpression of MIP2, a novel WD-repeat protein, promotes proliferation of H9c2 cells

International Nuclear Information System (INIS)

Wei, Xing; Song, Lan; Jiang, Lei; Wang, Guiliang; Luo, Xinjing; Zhang, Bin; Xiao, Xianzhong

2010-01-01

WD40 repeat proteins have a wide range of diverse biological functions including signal transduction, cell cycle regulation, RNA splicing, and transcription. Myocardial ischemic preconditioning up-regulated protein 2 (MIP2) is a novel member of the WD40 repeat proteins superfamily that contains five WD40 repeats. Little is known about its biological role, and the purpose of this study was to determine the role of MIP2 in regulating cellular proliferation. Transfection and constitutive expression of MIP2 in the rat cardiomyoblast cell line H9c2 results in enhanced growth of those cells as measured by cell number and is proportional to the amount of MIP2 expressed. Overexpression of MIP2 results in a shorter cell cycle, as measured by flow cytometry. Collectively, these data suggest that MIP2 may participate in the progression of cell proliferation in H9c2 cells.