Fitting Multimeric Protein Complexes into Electron Microscopy Maps Using 3D Zernike Descriptors

PubMed Central

Esquivel-Rodríguez, Juan; Kihara, Daisuke

2012-01-01

A novel computational method for fitting high-resolution structures of multiple proteins into a cryoelectron microscopy map is presented. The method named EMLZerD generates a pool of candidate multiple protein docking conformations of component proteins, which are later compared with a provided electron microscopy (EM) density map to select the ones that fit well into the EM map. The comparison of docking conformations and the EM map is performed using the 3D Zernike descriptor (3DZD), a mathematical series expansion of three-dimensional functions. The 3DZD provides a unified representation of the surface shape of multimeric protein complex models and EM maps, which allows a convenient, fast quantitative comparison of the three dimensional structural data. Out of 19 multimeric complexes tested, near native complex structures with a root mean square deviation of less than 2.5 Å were obtained for 14 cases while medium range resolution structures with correct topology were computed for the additional 5 cases. PMID:22417139

Fitting multimeric protein complexes into electron microscopy maps using 3D Zernike descriptors.

PubMed

Esquivel-Rodríguez, Juan; Kihara, Daisuke

2012-06-14

A novel computational method for fitting high-resolution structures of multiple proteins into a cryoelectron microscopy map is presented. The method named EMLZerD generates a pool of candidate multiple protein docking conformations of component proteins, which are later compared with a provided electron microscopy (EM) density map to select the ones that fit well into the EM map. The comparison of docking conformations and the EM map is performed using the 3D Zernike descriptor (3DZD), a mathematical series expansion of three-dimensional functions. The 3DZD provides a unified representation of the surface shape of multimeric protein complex models and EM maps, which allows a convenient, fast quantitative comparison of the three-dimensional structural data. Out of 19 multimeric complexes tested, near native complex structures with a root-mean-square deviation of less than 2.5 Å were obtained for 14 cases while medium range resolution structures with correct topology were computed for the additional 5 cases.

Cloning of murine RNA polymerase I-specific TAF factors: Conserved interactions between the subunits of the species-specific transcription initiation factor TIF-IB/SL1

PubMed Central

Heix, Jutta; Zomerdijk, Joost C. B. M.; Ravanpay, Ali; Tjian, Robert; Grummt, Ingrid

1997-01-01

Promoter selectivity for all three classes of eukaryotic RNA polymerases is brought about by multimeric protein complexes containing TATA box binding protein (TBP) and specific TBP-associated factors (TAFs). Unlike class II- and III-specific TBP–TAF complexes, the corresponding murine and human class I-specific transcription initiation factor TIF-IB/SL1 exhibits a pronounced selectivity for its homologous promoter. As a first step toward understanding the molecular basis of species-specific promoter recognition, we cloned the cDNAs encoding the three mouse pol I-specific TBP-associated factors (TAFIs) and compared the amino acid sequences of the murine TAFIs with their human counterparts. The four subunits from either species can form stable chimeric complexes that contain stoichiometric amounts of TBP and TAFIs, demonstrating that differences in the primary structure of human and mouse TAFIs do not dramatically alter the network of protein–protein contacts responsible for assembly of the multimeric complex. Thus, primate vs. rodent promoter selectivity mediated by the TBP–TAFI complex is likely to be the result of cumulative subtle differences between individual subunits that lead to species-specific properties of RNA polymerase I transcription. PMID:9050847

Electrostatic Forces as Dominant Interactions Between Proteins and Polyanions: an ESI MS Study of Fibroblast Growth Factor Binding to Heparin Oligomers

NASA Astrophysics Data System (ADS)

Minsky, Burcu Baykal; Dubin, Paul L.; Kaltashov, Igor A.

2017-04-01

The interactions between fibroblast growth factors (FGFs) and their receptors (FGFRs) are facilitated by heparan sulfate (HS) and heparin (Hp), highly sulfated biological polyelectrolytes. The molecular basis of FGF interactions with these polyelectrolytes is highly complex due to the structural heterogeneity of HS/Hp, and many details still remain elusive, especially the significance of charge density and minimal chain length of HS/Hp in growth factor recognition and multimerization. In this work, we use electrospray ionization mass spectrometry (ESI MS) to investigate the association of relatively homogeneous oligoheparins (octamer, dp8, and decamer, dp10) with acidic fibroblast growth factor (FGF-1). This growth factor forms 1:1, 2:1, and 3:1 protein/heparinoid complexes with both dp8 and dp10, and the fraction of bound protein is highly dependent on protein/heparinoid molar ratio. Multimeric complexes are preferentially formed on the highly sulfated Hp oligomers. Although a variety of oligomers appear to be binding-competent, there is a strong correlation between the affinity and the overall level of sulfation (the highest charge density polyanions binding FGF most strongly via multivalent interactions). These results show that the interactions between FGF-1 and Hp oligomers are primarily directed by electrostatics, and also demonstrate the power of ESI MS as a tool to study multiple binding equilibria between proteins and structurally heterogeneous polyanions.

Elastin-like polypeptide switches: A design strategy to detect multimeric proteins.

PubMed

Dhandhukia, Jugal P; Brill, Dab A; Kouhi, Aida; Pastuszka, Martha K; MacKay, J Andrew

2017-09-01

Elastin-Like Polypeptides (ELPs) reversibly phase separate in response to changes in temperature, pressure, concentration, pH, and ionic species. While powerful triggers, biological microenvironments present a multitude of more specific biological cues, such as antibodies, cytokines, and cell-surface receptors. To develop better biosensors and bioresponsive drug carriers, rational strategies are required to sense and respond to these target proteins. We recently reported that noncovalent association of two ELP fusion proteins to a "chemical inducer of dimerization" small molecule (1.5 kDa) induces phase separation at physiological temperatures. Having detected a small molecule, here we present the first evidence that ELP multimerization can also detect a much larger (60 kDa) protein target. To demonstrate this strategy, ELPs were biotinylated at their amino terminus and mixed with tetrameric streptavidin. At a stoichiometric ratio of [4:1], two to three biotin-ELPs associate with streptavidin into multimeric complexes with an apparent K d of 5 nM. The increased ELP density around a streptavidin core strongly promotes isothermal phase separation, which was tuned to occur at physiological temperature. This phase separation reverses upon saturation with excess streptavidin, which only favors [1:1] complexes. Together, these findings suggest that ELP association with multimeric biomolecules is a viable strategy to deliberately engineer ELPs that respond to multimeric protein substrates. © 2017 The Protein Society.

Dual Plug-and-Display Synthetic Assembly Using Orthogonal Reactive Proteins for Twin Antigen Immunization.

PubMed

Brune, Karl D; Buldun, Can M; Li, Yuanyuan; Taylor, Iona J; Brod, Florian; Biswas, Sumi; Howarth, Mark

2017-05-17

Engineering modular platforms to control biomolecular architecture can advance both the understanding and the manipulation of biological systems. Icosahedral particles uniformly displaying single antigens stimulate potent immune activation and have been successful in various licensed vaccines. However, it remains challenging to display multiple antigens on a single particle and to induce broader immunity protective across strains or even against distinct diseases. Here, we design a dually addressable synthetic nanoparticle by engineering the multimerizing coiled-coil IMX313 and two orthogonally reactive split proteins. SpyCatcher protein forms an isopeptide bond with SpyTag peptide through spontaneous amidation. SnoopCatcher forms an isopeptide bond with SnoopTag peptide through transamidation. SpyCatcher-IMX-SnoopCatcher provides a modular platform, whereby SpyTag-antigen and SnoopTag-antigen can be multimerized on opposite faces of the particle simply upon mixing. We demonstrate efficient derivatization of the platform with model proteins and complex pathogen-derived antigens. SpyCatcher-IMX-SnoopCatcher was expressed in Escherichia coli and was resilient to lyophilization or extreme temperatures. For the next generation of malaria vaccines, blocking the transmission of the parasite from human to mosquito is an important goal. SpyCatcher-IMX-SnoopCatcher multimerization of the leading transmission-blocking antigens Pfs25 and Pfs28 greatly enhanced the antibody response to both antigens in comparison to the monomeric proteins. This dual plug-and-display architecture should help to accelerate vaccine development for malaria and other diseases.

The Hexamer Structure of the Rift Valley Fever Virus Nucleoprotein Suggests a Mechanism for its Assembly into Ribonucleoprotein Complexes

PubMed Central

Ferron, François; Li, Zongli; Danek, Eric I.; Luo, Dahai; Wong, Yeehwa; Coutard, Bruno; Lantez, Violaine; Charrel, Rémi; Canard, Bruno; Walz, Thomas; Lescar, Julien

2011-01-01

Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs. PMID:21589902

Phosphatidylinositol 4,5-Bisphosphate Clusters the Cell Adhesion Molecule CD44 and Assembles a Specific CD44-Ezrin Heterocomplex, as Revealed by Small Angle Neutron Scattering*

PubMed Central

Chen, Xiaodong; Khajeh, Jahan Ali; Ju, Jeong Ho; Gupta, Yogesh K.; Stanley, Christopher B.; Do, Changwoo; Heller, William T.; Aggarwal, Aneel K.; Callaway, David J. E.; Bu, Zimei

2015-01-01

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes. PMID:25572402

Phosphatidylinositol 4,5-bisphosphate clusters the cell adhesion molecule CD44 and assembles a specific CD44-Ezrin heterocomplex, as revealed by small angle neutron scattering.

PubMed

Chen, Xiaodong; Khajeh, Jahan Ali; Ju, Jeong Ho; Gupta, Yogesh K; Stanley, Christopher B; Do, Changwoo; Heller, William T; Aggarwal, Aneel K; Callaway, David J E; Bu, Zimei

2015-03-06

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Deficiency of PHB complex impairs respiratory supercomplex formation and activates mitochondrial flashes.

PubMed

Jian, Chongshu; Xu, Fengli; Hou, Tingting; Sun, Tao; Li, Jinghang; Cheng, Heping; Wang, Xianhua

2017-08-01

Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins. PHBs form multimeric ring complexes acting as scaffolds in the inner mitochondrial membrane. Mitochondrial flashes (mitoflashes) are newly discovered mitochondrial signaling events that reflect electrical and chemical excitations of the organelle. Here, we investigate the possible roles of PHBs in the regulation of mitoflash signaling. Downregulation of PHBs increases mitoflash frequency by up to 5.4-fold due to elevated basal reactive oxygen species (ROS) production in the mitochondria. Mechanistically, PHB deficiency impairs the formation of mitochondrial respiratory supercomplexes (RSCs) without altering the abundance of individual respiratory complex subunits. These impairments induced by PHB deficiency are effectively rescued by co-expression of PHB1 and PHB2, indicating that the multimeric PHB complex acts as the functional unit. Furthermore, downregulating other RSC assembly factors, including SCAFI (also known as COX7A2L), RCF1a (HIGD1A), RCF1b (HIGD2A), UQCC3 and SLP2 (STOML2), all activate mitoflashes through elevating mitochondrial ROS production. Our findings identify the PHB complex as a new regulator of RSC formation and mitoflash signaling, and delineate a general relationship among RSC formation, basal ROS production and mitoflash biogenesis. © 2017. Published by The Company of Biologists Ltd.

Multimeric complexes among ankyrin-repeat and SOCS-box protein 9 (ASB9), ElonginBC, and Cullin 5: insights into the structure and assembly of ECS-type Cullin-RING E3 ubiquitin ligases.

PubMed

Thomas, Jemima C; Matak-Vinkovic, Dijana; Van Molle, Inge; Ciulli, Alessio

2013-08-06

Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM-MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9-EloBC-Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM-MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.

Multimeric Complexes among Ankyrin-Repeat and SOCS-box Protein 9 (ASB9), ElonginBC, and Cullin 5: Insights into the Structure and Assembly of ECS-type Cullin-RING E3 Ubiquitin Ligases

PubMed Central

2013-01-01

Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC–Cullin–SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM–MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9–EloBC–Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM–MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems. PMID:23837592

Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.

PubMed

Harding, Peter J; Attrill, Helen; Boehringer, Jonas; Ross, Simon; Wadhams, George H; Smith, Eleanor; Armitage, Judith P; Watts, Anthony

2009-02-01

Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.

The disaggregation theory of signal transduction revisited: further evidence that G proteins are multimeric and disaggregate to monomers when activated.

PubMed

Jahangeer, S; Rodbell, M

1993-10-01

We have compared the sedimentation rates on sucrose gradients of the heterotrimeric GTP-binding regulatory (G) proteins Gs, G(o), Gi, and Gq extracted from rat brain synaptoneurosomes with Lubrol and digitonin. The individual alpha and beta subunits were monitored with specific antisera. In all cases, both subunits cosedimented, indicating that the subunits are likely complexed as heterotrimers. When extracted with Lubrol all of the G proteins sedimented with rates of about 4.5 S (consistent with heterotrimers) whereas digitonin extracted 60% of the G proteins with peaks at 11 S; 40% pelleted as larger structures. Digitonin-extracted Gi was cross-linked by p-phenylenedimaleimide, yielding structures too large to enter polyacrylamide gels. No cross-linking of Lubrol-extracted Gi occurred. Treatment of the membranes with guanosine 5'-[gamma-thio]triphosphate and Mg2+ yielded digitonin-extracted structures with peak sedimentation values of 8.5 S--i.e., comparable to that of purified G(o) in digitonin and considerably larger than the Lubrol-extracted 2S structures representing the separated alpha and beta gamma subunits formed by the actions of guanosine 5'-[gamma-thio]triphosphate. It is concluded that the multimeric structures of G proteins in brain membranes are at least partially preserved in digitonin and that activation of these structures in membranes yields monomers of G proteins rather than the disaggregated products (alpha and beta gamma complexes) observed in Lubrol. It is proposed that hormones and GTP affect the dynamic interplay between multimeric G proteins and receptors in a fashion analogous to the actions of ATP on the dynamic interactions between myosin and actin filaments. Signal transduction is mediated by activated monomers released from the multimers during the activation process.

The disaggregation theory of signal transduction revisited: further evidence that G proteins are multimeric and disaggregate to monomers when activated.

PubMed Central

Jahangeer, S; Rodbell, M

1993-01-01

We have compared the sedimentation rates on sucrose gradients of the heterotrimeric GTP-binding regulatory (G) proteins Gs, G(o), Gi, and Gq extracted from rat brain synaptoneurosomes with Lubrol and digitonin. The individual alpha and beta subunits were monitored with specific antisera. In all cases, both subunits cosedimented, indicating that the subunits are likely complexed as heterotrimers. When extracted with Lubrol all of the G proteins sedimented with rates of about 4.5 S (consistent with heterotrimers) whereas digitonin extracted 60% of the G proteins with peaks at 11 S; 40% pelleted as larger structures. Digitonin-extracted Gi was cross-linked by p-phenylenedimaleimide, yielding structures too large to enter polyacrylamide gels. No cross-linking of Lubrol-extracted Gi occurred. Treatment of the membranes with guanosine 5'-[gamma-thio]triphosphate and Mg2+ yielded digitonin-extracted structures with peak sedimentation values of 8.5 S--i.e., comparable to that of purified G(o) in digitonin and considerably larger than the Lubrol-extracted 2S structures representing the separated alpha and beta gamma subunits formed by the actions of guanosine 5'-[gamma-thio]triphosphate. It is concluded that the multimeric structures of G proteins in brain membranes are at least partially preserved in digitonin and that activation of these structures in membranes yields monomers of G proteins rather than the disaggregated products (alpha and beta gamma complexes) observed in Lubrol. It is proposed that hormones and GTP affect the dynamic interplay between multimeric G proteins and receptors in a fashion analogous to the actions of ATP on the dynamic interactions between myosin and actin filaments. Signal transduction is mediated by activated monomers released from the multimers during the activation process. Images Fig. 1 Fig. 2 PMID:8415607

Design and Testing of a Thermostable Platform for Multimerization of Single Domain Antibodies

DTIC Science & Technology

2012-08-01

1 DESIGN AND TESTING OF A THERMOSTABLE PLATFORM FOR MULTIMERIZATION OF SINGLE DOMAIN ANTIBODIES ECBC-TR...Army position unless so designated by other authorizing documents. REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704-0188 Public...ADDRESS. 1. REPORT DATE (DD-MM-YYYY) XX-08-2012 2. REPORT TYPE Final 3. DATES COVERED (From - To) Oct 2008 - Sep 2010 4. TITLE AND SUBTITLE Design

The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

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

Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.

Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

DOE PAGES

Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.; ...

2014-12-08

Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

An RNA-Binding Multimer Specifies Nematode Sperm Fate.

PubMed

Aoki, Scott T; Porter, Douglas F; Prasad, Aman; Wickens, Marvin; Bingman, Craig A; Kimble, Judith

2018-06-26

FOG-3 is a master regulator of sperm fate in Caenorhabditis elegans and homologous to Tob/BTG proteins, which in mammals are monomeric adaptors that recruit enzymes to RNA binding proteins. Here, we determine the FOG-3 crystal structure and in vitro demonstrate that FOG-3 forms dimers that can multimerize. The FOG-3 multimeric structure has a basic surface potential, suggestive of binding nucleic acid. Consistent with that prediction, FOG-3 binds directly to nearly 1,000 RNAs in nematode spermatogenic germ cells. Most binding is to the 3' UTR, and most targets (94%) are oogenic mRNAs, even though assayed in spermatogenic cells. When tethered to a reporter mRNA, FOG-3 represses its expression. Together these findings elucidate the molecular mechanism of sperm fate specification and reveal the evolution of a protein from monomeric to multimeric form with acquisition of a distinct mode of mRNA repression. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

High Molecular Weight Forms of Mammalian Respiratory Chain Complex II

PubMed Central

Nůsková, Hana; Holzerová, Eliška; Vrbacký, Marek; Pecina, Petr; Hejzlarová, Kateřina; Kľučková, Katarína; Rohlena, Jakub; Neuzil, Jiri; Houštěk, Josef

2013-01-01

Mitochondrial respiratory chain is organised into supramolecular structures that can be preserved in mild detergent solubilisates and resolved by native electrophoretic systems. Supercomplexes of respiratory complexes I, III and IV as well as multimeric forms of ATP synthase are well established. However, the involvement of complex II, linking respiratory chain with tricarboxylic acid cycle, in mitochondrial supercomplexes is questionable. Here we show that digitonin-solubilised complex II quantitatively forms high molecular weight structures (CIIhmw) that can be resolved by clear native electrophoresis. CIIhmw structures are enzymatically active and differ in electrophoretic mobility between tissues (500 – over 1000 kDa) and cultured cells (400–670 kDa). While their formation is unaffected by isolated defects in other respiratory chain complexes, they are destabilised in mtDNA-depleted, rho0 cells. Molecular interactions responsible for the assembly of CIIhmw are rather weak with the complexes being more stable in tissues than in cultured cells. While electrophoretic studies and immunoprecipitation experiments of CIIhmw do not indicate specific interactions with the respiratory chain complexes I, III or IV or enzymes of the tricarboxylic acid cycle, they point out to a specific interaction between CII and ATP synthase. PMID:23967256

Characterization of Hepatitis C Virus Core Protein Multimerization and Membrane Envelopment: Revelation of a Cascade of Core-Membrane Interactions ▿

PubMed Central

Ai, Li-Shuang; Lee, Yu-Wen; Chen, Steve S.-L.

2009-01-01

The molecular basis underlying hepatitis C virus (HCV) core protein maturation and morphogenesis remains elusive. We characterized the concerted events associated with core protein multimerization and interaction with membranes. Analyses of core proteins expressed from a subgenomic system showed that the signal sequence located between the core and envelope glycoprotein E1 is critical for core association with endoplasmic reticula (ER)/late endosomes and the core's envelopment by membranes, which was judged by the core's acquisition of resistance to proteinase K digestion. Despite exerting an inhibitory effect on the core's association with membranes, (Z-LL)2-ketone, a specific inhibitor of signal peptide peptidase (SPP), did not affect core multimeric complex formation, suggesting that oligomeric core complex formation proceeds prior to or upon core attachment to membranes. Protease-resistant core complexes that contained both innate and processed proteins were detected in the presence of (Z-LL)2-ketone, implying that core envelopment occurs after intramembrane cleavage. Mutations of the core that prevent signal peptide cleavage or coexpression with an SPP loss-of-function D219A mutant decreased the core's envelopment, demonstrating that SPP-mediated cleavage is required for core envelopment. Analyses of core mutants with a deletion in domain I revealed that this domain contains sequences crucial for core envelopment. The core proteins expressed by infectious JFH1 and Jc1 RNAs in Huh7 cells also assembled into a multimeric complex, associated with ER/late-endosomal membranes, and were enveloped by membranes. Treatment with (Z-LL)2-ketone or coexpression with D219A mutant SPP interfered with both core envelopment and infectious HCV production, indicating a critical role of core envelopment in HCV morphogenesis. The results provide mechanistic insights into the sequential and coordinated processes during the association of the HCV core protein with membranes in the early phase of virus maturation and morphogenesis. PMID:19605478

Dishevelled phosphorylation, subcellular localization and multimerization regulate its role in early embryogenesis

PubMed Central

Rothbächer, Ute; Laurent, Micheline N.; Deardorff, Matthew A.; Klein, Peter S.; Cho, Ken W.Y.; Fraser, Scott E.

2000-01-01

Dishevelled (Dsh) induces a secondary axis and can translocate to the membrane when activated by Frizzleds; however, dominant-negative approaches have not supported a role for Dsh in primary axis formation. We demonstrate that the Dsh protein is post-translationally modified at the dorsal side of the embryo: timing and position of this regulation suggests a role of Dsh in dorsal–ventral patterning in Xenopus. To create functional links between these properties of Dsh we analyzed the influence of endogenous Frizzleds and the Dsh domain dependency for these characteristics. Xenopus Frizzleds phosphorylate and translocate Xdsh to the membrane irrespective of their differential ectopic axes inducing abilities, showing that translocation is insufficient for axis induction. Dsh deletion analysis revealed that axis inducing abilities did not segregate with Xdsh membrane association. The DIX region and a short stretch at the N–terminus of the DEP domain are necessary for axis induction while the DEP region is required for Dsh membrane association and its phosphorylation. In addition, Dsh forms homomeric complexes in embryos suggesting that multimerization is important for its proper function. PMID:10698942

Identification of a probable pore-forming domain in the multimeric vacuolar anion channel AtALMT9.

PubMed

Zhang, Jingbo; Baetz, Ulrike; Krügel, Undine; Martinoia, Enrico; De Angeli, Alexis

2013-10-01

Aluminum-activated malate transporters (ALMTs) form an important family of anion channels involved in fundamental physiological processes in plants. Because of their importance, the role of ALMTs in plant physiology is studied extensively. In contrast, the structural basis of their functional properties is largely unknown. This lack of information limits the understanding of the functional and physiological differences between ALMTs and their impact on anion transport in plants. This study aimed at investigating the structural organization of the transmembrane domain of the Arabidopsis (Arabidopsis thaliana) vacuolar channel AtALMT9. For that purpose, we performed a large-scale mutagenesis analysis and found two residues that form a salt bridge between the first and second putative transmembrane α-helices (TMα1 and TMα2). Furthermore, using a combination of pharmacological and mutagenesis approaches, we identified citrate as an "open channel blocker" of AtALMT9 and used this tool to examine the inhibition sensitivity of different point mutants of highly conserved amino acid residues. By this means, we found a stretch within the cytosolic moiety of the TMα5 that is a probable pore-forming domain. Moreover, using a citrate-insensitive AtALMT9 mutant and biochemical approaches, we could demonstrate that AtALMT9 forms a multimeric complex that is supposedly composed of four subunits. In summary, our data provide, to our knowledge, the first evidence about the structural organization of an ion channel of the ALMT family. We suggest that AtALMT9 is a tetramer and that the TMα5 domains of the subunits contribute to form the pore of this anion channel.

Syntrophomonas wolfei Uses an NADH-Dependent, Ferredoxin-Independent [FeFe]-Hydrogenase To Reoxidize NADH

PubMed Central

Losey, Nathaniel A.; Mus, Florence; Peters, John W.; Le, Huynh M.

2017-01-01

ABSTRACT Syntrophomonas wolfei syntrophically oxidizes short-chain fatty acids (four to eight carbons in length) when grown in coculture with a hydrogen- and/or formate-using methanogen. The oxidation of 3-hydroxybutyryl-coenzyme A (CoA), formed during butyrate metabolism, results in the production of NADH. The enzyme systems involved in NADH reoxidation in S. wolfei are not well understood. The genome of S. wolfei contains a multimeric [FeFe]-hydrogenase that may be a mechanism for NADH reoxidation. The S. wolfei genes for the multimeric [FeFe]-hydrogenase (hyd1ABC; SWOL_RS05165, SWOL_RS05170, SWOL_RS05175) and [FeFe]-hydrogenase maturation proteins (SWOL_RS05180, SWOL_RS05190, SWOL_RS01625) were coexpressed in Escherichia coli, and the recombinant Hyd1ABC was purified and characterized. The purified recombinant Hyd1ABC was a heterotrimer with an αβγ configuration and a molecular mass of 115 kDa. Hyd1ABC contained 29.2 ± 1.49 mol of Fe and 0.7 mol of flavin mononucleotide (FMN) per mole enzyme. The purified, recombinant Hyd1ABC reduced NAD+ and oxidized NADH without the presence of ferredoxin. The HydB subunit of the S. wolfei multimeric [FeFe]-hydrogenase lacks two iron-sulfur centers that are present in known confurcating NADH- and ferredoxin-dependent [FeFe]-hydrogenases. Hyd1ABC is a NADH-dependent hydrogenase that produces hydrogen from NADH without the need of reduced ferredoxin, which differs from confurcating [FeFe]-hydrogenases. Hyd1ABC provides a mechanism by which S. wolfei can reoxidize NADH produced during syntrophic butyrate oxidation when low hydrogen partial pressures are maintained by a hydrogen-consuming microorganism. IMPORTANCE Our work provides mechanistic understanding of the obligate metabolic coupling that occurs between hydrogen-producing fatty and aromatic acid-degrading microorganisms and their hydrogen-consuming partners in the process called syntrophy (feeding together). The multimeric [FeFe]-hydrogenase used NADH without the involvement of reduced ferredoxin. The multimeric [FeFe]-hydrogenase would produce hydrogen from NADH only when hydrogen concentrations were low. Hydrogen production from NADH by Syntrophomonas wolfei would likely cease before any detectable amount of cell growth occurred. Thus, continual hydrogen production requires the presence of a hydrogen-consuming partner to keep hydrogen concentrations low and explains, in part, the obligate requirement that S. wolfei has for a hydrogen-consuming partner organism during growth on butyrate. We have successfully expressed genes encoding a multimeric [FeFe]-hydrogenase in E. coli, demonstrating that such an approach can be advantageous to characterize complex redox proteins from difficult-to-culture microorganisms. PMID:28802265

Psp Stress Response Proteins Form a Complex with Mislocalized Secretins in the Yersinia enterocolitica Cytoplasmic Membrane.

PubMed

Srivastava, Disha; Moumene, Amal; Flores-Kim, Josué; Darwin, Andrew J

2017-09-12

The bacterial phage shock protein system (Psp) is a conserved extracytoplasmic stress response that is essential for the virulence of some pathogens, including Yersinia enterocolitica It is induced by events that can compromise inner membrane (IM) integrity, including the mislocalization of outer membrane pore-forming proteins called secretins. In the absence of the Psp system, secretin mislocalization permeabilizes the IM and causes rapid cell death. The Psp proteins PspB and PspC form an integral IM complex with two independent roles. First, the PspBC complex is required to activate the Psp response in response to some inducing triggers, including a mislocalized secretin. Second, PspBC are sufficient to counteract mislocalized secretin toxicity. Remarkably, secretin mislocalization into the IM induces psp gene expression without significantly affecting the expression of any other genes. Furthermore, psp null strains are killed by mislocalized secretins, whereas no other null mutants have been found to share this specific secretin sensitivity. This suggests an exquisitely specific relationship between secretins and the Psp system, but there has been no mechanism described to explain this. In this study, we addressed this deficiency by using a coimmunoprecipitation approach to show that the Psp proteins form a specific complex with mislocalized secretins in the Y. enterocolitica IM. Importantly, analysis of different secretin mutant proteins also revealed that this interaction is absolutely dependent on a secretin adopting a multimeric state. Therefore, the Psp system has evolved with the ability to detect and detoxify dangerous secretin multimers while ignoring the presence of innocuous monomers. IMPORTANCE The phage shock protein (Psp) response has been linked to important phenotypes in diverse bacteria, including those related to antibiotic resistance, biofilm formation, and virulence. This has generated widespread interest in understanding various aspects of its function. Outer membrane secretin proteins are essential components of export systems required for the virulence of many bacterial pathogens. However, secretins can mislocalize into the inner membrane, and this induces the Psp response in a highly specific manner and kills Psp-defective strains with similar specificity. There has been no mechanism described to explain this exquisitely specific relationship between secretins and the Psp system. Therefore, this study provides a critical advance by discovering that Psp effector proteins form a complex with secretins in the Yersinia enterocolitica inner membrane. Remarkably, this interaction is absolutely dependent on a secretin adopting its multimeric state. Therefore, the Psp system detects and detoxifies dangerous secretin multimers, while ignoring the presence of innocuous secretin monomers. Copyright © 2017 Srivastava et al.

Dissociation of glucocerebrosidase dimer in solution by its co-factor, saposin C

DOE PAGES

Gruschus, James M.; Jiang, Zhiping; Yap, Thai Leong; ...

2015-01-16

Mutations in the gene for the lysosomal enzyme glucocerebrosidase (GCase) cause Gaucher disease and are the most common risk factor for Parkinson disease (PD). Analytical ultracentrifugation of 8 μM GCase shows equilibrium between monomer and dimer forms. However, in the presence of its co-factor saposin C (Sap C), only monomer GCase is seen. Isothermal calorimetry confirms that Sap C associates with GCase in solution in a 1:1 complex (K d = 2.1 ± 1.1 μM). Saturation cross-transfer NMR determined that the region of Sap C contacting GCase includes residues 63–66 and 74–76, which is distinct from the region known tomore » enhance GCase activity. Because α-synuclein (α-syn), a protein closely associated with PD etiology, competes with Sap C for GCase binding, its interaction with GCase was also measured by ultracentrifugation and saturation cross-transfer. Unlike Sap C, binding of α-syn to GCase does not affect multimerization. However, adding α-syn reduces saturation cross-transfer from Sap C to GCase, confirming displacement. To explore where Sap C might disrupt multimeric GCase, GCase x-ray structures were analyzed using the program PISA, which predicted stable dimer and tetramer forms. In conclusion, for the most frequently predicted multimer interface, the GCase active sites are partially buried, suggesting that Sap C might disrupt the multimer by binding near the active site.« less

A Tyrosine Residue on the TSH Receptor Stabilizes Multimer Formation

PubMed Central

Latif, Rauf; Michalek, Krzysztof; Morshed, Syed Ahmed; Davies, Terry F.

2010-01-01

Background The thyrotropin stimulating hormone receptor (TSHR) is a G protein coupled receptor (GPCR) with a large ectodomain. The ligand, TSH, acting via this receptor regulates thyroid growth and thyroid hormone production and secretion. The TSH receptor (TSHR) undergoes complex post –translational modifications including intramolecular cleavage and receptor multimerization. Since monomeric and multimeric receptors coexist in cells, understanding the functional role of just the TSHR multimers is difficult. Therefore, to help understand the physiological significance of receptor multimerization, it will be necessary to abrogate multimer formation, which requires identifying the ectodomain and endodomain interaction sites on the TSHR. Here, we have examined the contribution of the ectodomain to constitutive multimerization of the TSHR and determined the possible residue(s) that may be involved in this interaction. Methodology/Principal Findings We studied ectodomain multimer formation by expressing the extracellular domain of the TSHR linked to a glycophosphotidyl (GPI) anchor in both stable and transient expression systems. Using co-immunoprecipitation and FRET of tagged receptors, we established that the TSH receptor ectodomain was capable of multimerization even when totally devoid of the transmembrane domain. Further, we studied the effect of two residues that likely made critical contact points in this interaction. We showed that a conserved tyrosine residue (Y116) on the convex surface of the LRR3 was a critical residue in ectodomain multimer formation since mutation of this residue to serine totally abrogated ectodomain multimers. This abrogation was not seen with the mutation of cysteine 176 on the inner side of the LRR5, demonstrating that inter-receptor disulfide bonding was not involved in ectodomain multimer formation. Additionally, the Y116 mutation in the intact wild type receptor enhanced receptor degradation. Conclusions/Significance These data establish the TSH receptor ectodomain as one site of multimerization, independent of the transmembrane region, and that this interaction was primarily via a conserved tyrosine residue in LRR3. PMID:20195479

A novel molecular dynamics approach to evaluate the effect of phosphorylation on multimeric protein interface: the αB-Crystallin case study.

PubMed

Chiappori, Federica; Mattiazzi, Luca; Milanesi, Luciano; Merelli, Ivan

2016-03-02

Phosphorylation is one of the most important post-translational modifications (PTM) employed by cells to regulate several cellular processes. Studying the effects of phosphorylations on protein structures allows to investigate the modulation mechanisms of several proteins including chaperones, like the small HSPs, which display different multimeric structures according to the phosphorylation of a few serine residues. In this context, the proposed study is aimed at finding a method to correlate different PTM patterns (in particular phosphorylations at the monomers interface of multimeric complexes) with the dynamic behaviour of the complex, using physicochemical parameters derived from molecular dynamics simulations in the timescale of nanoseconds. We have developed a methodology relying on computing nine physicochemical parameters, derived from the analysis of short MD simulations, and combined with N identifiers that characterize the PTMs of the analysed protein. The nine general parameters were validated on three proteins, with known post-translational modified conformation and unmodified conformation. Then, we applied this approach to the case study of αB-Crystallin, a chaperone which multimeric state (up to 40 units) is supposed to be controlled by phosphorylation of Ser45 and Ser59. Phosphorylation of serines at the dimer interface induces the release of hexamers, the active state of αB-Crystallin. 30 ns of MD simulation were obtained for each possible combination of dimer phosphorylation state and average values of structural, dynamic, energetic and functional features were calculated on the equilibrated portion of the trajectories. Principal Component Analysis was applied to the parameters and the first five Principal Components, which summed up to 84 % of the total variance, were finally considered. The validation of this approach on multimeric proteins, which structures were known both modified and unmodified, allowed us to propose a new approach that can be used to predict the impact of PTM patterns in multi-modified proteins using data collected from short molecular dynamics simulations. Analysis on the αB-Crystallin case study clusters together all-P dimers with all-P hexamers and no-P dimer with no-P hexamer and results suggest a great influence of Ser59 phosphorylation on chain B.

Role of carbohydrate in multimeric structure of factor VIII/von Willebrand factor protein.

PubMed Central

Gralnick, H R; Williams, S B; Rick, M E

1983-01-01

The carbohydrate moiety of the factor VIII/von Willebrand (vW) factor protein is important in the expression of vW factor activity and the intravascular survival of the protein. Studies of normal human factor VIII/vW factor protein indicate that there is a requirement of a full complement of penultimate galactose for the maintenance of a normal multimeric structure. Release of penultimate galactose by beta-galactosidase or modification by galactose oxidase results in loss of the largest molecular weight multimers and increased numbers of intermediate and smaller multimers. In contrast, terminal galactose on the factor VIII/vW factor protein does not appear to play a significant role in the maintenance of the multimer organization. The abnormalities in multimeric structure and molecular size were demonstrated by NaDodSO4/polyacrylamide/agarose gel electrophoresis, NaDodSO4/glyoxyl-agarose electrophoresis, and sucrose density ultracentrifugation. These studies indicate that the penultimate galactose plays a role in the maintenance of the largest multimers of the factor VIII/vW factor protein. This may explain why, in some patients with variant forms of vW disease, a carbohydrate abnormality also may affect the multimeric structure of the plasma factor VIII/vW factor protein. Images PMID:6601805

Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor.

PubMed

Jouvin, M H; Adamczewski, M; Numerof, R; Letourneur, O; Vallé, A; Kinet, J P

1994-02-25

Nonreceptor tyrosine kinases such as the newly described 70-kDa (ZAP-70/Syk) and Src-related tyrosine kinases are coupled to a variety of receptors, including the antigen receptors on B- and T-cells and the Fc receptors for IgE (Fc epsilon RI) and IgG (Fc gamma RI, Fc gamma RIII/CD16). Various subunits of these receptors contain homologous activation motifs which appear capable of autonomously triggering cell activation. Two forms of this motif are present in the Fc epsilon RI multimeric complex: one in the beta chain and one in the gamma chain. Here we show that each of the two tyrosine kinases known to be involved in Fc epsilon RI signaling is controlled by a distinct motif-containing chain. Lyn associates with the nonactivated beta chain, whereas gamma promotes the activation of Syk. We also show that neither the beta nor the gamma motif alone can account for the full signaling capacity of the entire receptor. We propose that, upon triggering of the tetrameric receptor, Lyn already bound to beta becomes activated and phosphorylates beta and gamma; the phosphorylation of gamma induces the association of Syk with gamma and also the activation of Syk, resulting in the phosphorylation and activation of phospholipase C gamma 1. Cooperative recruitment of specific kinases by the various signaling chains found in this family of antigen receptors could represent a way to achieve the full signaling capacity of the multimeric complexes.

A Rhizavidin Monomer with Nearly Multimeric Avidin-Like Binding Stability Against Biotin Conjugates.

PubMed

Lee, Jeong Min; Kim, Jung A; Yen, Tzu-Chi; Lee, In Hwan; Ahn, Byungjun; Lee, Younghoon; Hsieh, Chia-Lung; Kim, Ho Min; Jung, Yongwon

2016-03-01

Developing a monomeric form of an avidin-like protein with highly stable biotin binding properties has been a major challenge in biotin-avidin linking technology. Here we report a monomeric avidin-like protein-enhanced monoavidin-with off-rates almost comparable to those of multimeric avidin proteins against various biotin conjugates. Enhanced monoavidin (eMA) was developed from naturally dimeric rhizavidin by optimally maintaining protein rigidity during monomerization and additionally shielding the bound biotin by diverse engineering of the surface residues. eMA allowed the monovalent and nonperturbing labeling of head-group-biotinylated lipids in bilayer membranes. In addition, we fabricated an unprecedented 24-meric avidin probe by fusing eMA to a multimeric cage protein. The 24-meric avidin and eMA were utilized to demonstrate how artificial clustering of cell-surface proteins greatly enhances the internalization rates of assembled proteins on live cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane

PubMed Central

Richardson, Lynn G. L.; Paila, Yamuna D.; Siman, Steven R.; Chen, Yi; Smith, Matthew D.; Schnell, Danny J.

2014-01-01

The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β–barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus. PMID:24966864

Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane.

PubMed

Richardson, Lynn G L; Paila, Yamuna D; Siman, Steven R; Chen, Yi; Smith, Matthew D; Schnell, Danny J

2014-01-01

The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β-barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export.

PubMed

Behrens, Ryan T; Aligeti, Mounavya; Pocock, Ginger M; Higgins, Christina A; Sherer, Nathan M

2017-02-01

HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding. HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place. Copyright © 2017 American Society for Microbiology.

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

PubMed Central

Behrens, Ryan T.; Aligeti, Mounavya; Pocock, Ginger M.; Higgins, Christina A.

2016-01-01

ABSTRACT HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding. IMPORTANCE HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place. PMID:27852860

A conservation and biophysics guided stochastic approach to refining docked multimeric proteins.

PubMed

Akbal-Delibas, Bahar; Haspel, Nurit

2013-01-01

We introduce a protein docking refinement method that accepts complexes consisting of any number of monomeric units. The method uses a scoring function based on a tight coupling between evolutionary conservation, geometry and physico-chemical interactions. Understanding the role of protein complexes in the basic biology of organisms heavily relies on the detection of protein complexes and their structures. Different computational docking methods are developed for this purpose, however, these methods are often not accurate and their results need to be further refined to improve the geometry and the energy of the resulting complexes. Also, despite the fact that complexes in nature often have more than two monomers, most docking methods focus on dimers since the computational complexity increases exponentially due to the addition of monomeric units. Our results show that the refinement scheme can efficiently handle complexes with more than two monomers by biasing the results towards complexes with native interactions, filtering out false positive results. Our refined complexes have better IRMSDs with respect to the known complexes and lower energies than those initial docked structures. Evolutionary conservation information allows us to bias our results towards possible functional interfaces, and the probabilistic selection scheme helps us to escape local energy minima. We aim to incorporate our refinement method in a larger framework which also enables docking of multimeric complexes given only monomeric structures.

Hu antigen R (HuR) multimerization contributes to glioma disease progression.

PubMed

Filippova, Natalia; Yang, Xiuhua; Ananthan, Subramaniam; Sorochinsky, Anastasia; Hackney, James R; Gentry, Zachery; Bae, Sejong; King, Peter; Nabors, L Burt

2017-10-13

Among primary brain cancers, gliomas are the most deadly and most refractory to current treatment modalities. Previous reports overwhelmingly support the role of the RNA-binding protein Hu antigen R (HuR) as a positive regulator of glioma disease progression. HuR expression is consistently elevated in tumor tissues, and a cytoplasmic localization appears essential for HuR-dependent oncogenic transformation. Here, we report HuR aggregation (multimerization) in glioma and the analysis of this tumor-specific HuR protein multimerization in clinical brain tumor samples. Using a split luciferase assay, a bioluminescence resonance energy transfer technique, and site-directed mutagenesis, we examined the domains involved in HuR multimerization. Results obtained with the combination of the split HuR luciferase assay with the bioluminescence resonance energy transfer technique suggested that multiple (at least three) HuR molecules come together during HuR multimerization in glioma cells. Using these data, we developed a model of HuR multimerization in glioma cells. We also demonstrate that exposing glioma cells to the HuR inhibitor tanshinone group compound 15,16-dihydrotanshinone-I or to the newly identified compound 5 disrupts HuR multimerization modules and reduces tumor cell survival and proliferation. In summary, our findings provide new insights into HuR multimerization in glioma and highlight possible pharmacological approaches for targeting HuR domains involved in cancer cell-specific multimerization.

Stoichiometry for binding and transport by the twin arginine translocation system.

PubMed

Celedon, Jose M; Cline, Kenneth

2012-05-14

Twin arginine translocation (Tat) systems transport large folded proteins across sealed membranes. Tat systems accomplish this feat with three membrane components organized in two complexes. In thylakoid membranes, cpTatC and Hcf106 comprise a large receptor complex containing an estimated eight cpTatC-Hcf106 pairs. Protein transport occurs when Tha4 joins the receptor complex as an oligomer of uncertain size that is thought to form the protein-conducting structure. Here, binding analyses with intact membranes or purified complexes indicate that each receptor complex could bind eight precursor proteins. Kinetic analysis of translocation showed that each precursor-bound site was independently functional for transport, and, with sufficient Tha4, all sites were concurrently active for transport. Tha4 titration determined that ∼26 Tha4 protomers were required for transport of each OE17 (oxygen-evolving complex subunit of 17 kD) precursor protein. Our results suggest that, when fully saturated with precursor proteins and Tha4, the Tat translocase is an ∼2.2-megadalton complex that can individually transport eight precursor proteins or cooperatively transport multimeric precursors.

Sonic hedgehog multimerization: a self-organizing event driven by post-translational modifications?

PubMed

Koleva, Mirella V; Rothery, Stephen; Spitaler, Martin; Neil, Mark A A; Magee, Anthony I

2015-01-01

Sonic hedgehog (Shh) is a morphogen active during vertebrate development and tissue homeostasis in adulthood. Dysregulation of the Shh signalling pathway is known to incite carcinogenesis. Due to the highly lipophilic nature of this protein imparted by two post-translational modifications, Shh's method of transit through the aqueous extracellular milieu has been a long-standing conundrum, prompting the proposition of numerous hypotheses to explain the manner of its displacement from the surface of the producing cell. Detection of high molecular-weight complexes of Shh in the intercellular environment has indicated that the protein achieves this by accumulating into multimeric structures prior to release from producing cells. The mechanism of assembly of the multimers, however, has hitherto remained mysterious and contentious. Here, with the aid of high-resolution optical imaging and post-translational modification mutants of Shh, we show that the C-terminal cholesterol and the N-terminal palmitate adducts contribute to the assembly of large multimers and regulate their shape. Moreover, we show that small Shh multimers are produced in the absence of any lipid modifications. Based on an assessment of the distribution of various dimensional characteristics of individual Shh clusters, in parallel with deductions about the kinetics of release of the protein from the producing cells, we conclude that multimerization is driven by self-assembly underpinned by the law of mass action. We speculate that the lipid modifications augment the size of the multimolecular complexes through prolonging their association with the exoplasmic membrane.

TIA-1 Self-Multimerization, Phase Separation, and Recruitment into Stress Granules Are Dynamically Regulated by Zn2.

PubMed

Rayman, Joseph B; Karl, Kevin A; Kandel, Eric R

2018-01-02

Stress granules are non-membranous structures that transiently form in the cytoplasm during cellular stress, where they promote translational repression of non-essential RNAs and modulate cell signaling by sequestering key signal transduction proteins. These and other functions of stress granules facilitate an adaptive cellular response to environmental adversity. A key component of stress granules is the prion-related RNA-binding protein, T cell intracellular antigen-1 (TIA-1). Here, we report that recombinant TIA-1 undergoes rapid multimerization and phase separation in the presence of divalent zinc, which can be reversed by the zinc chelator, TPEN. Similarly, the formation and maintenance of TIA-1-positive stress granules in arsenite-treated cells are inhibited by TPEN. In addition, Zn 2+ is released in cells treated with arsenite, before stress granule formation. These findings suggest that Zn 2+ is a physiological ligand of TIA-1, acting as a stress-inducible second messenger to promote multimerization of TIA-1 and subsequent localization into stress granules. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Mammalian Otolin: A Multimeric Glycoprotein Specific to the Inner Ear that Interacts with Otoconial Matrix Protein Otoconin-90 and Cerebellin-1

PubMed Central

Deans, Michael R.; Peterson, Jonathan M.; Wong, G. William

2010-01-01

Background The mammalian otoconial membrane is a dense extracellular matrix containing bio-mineralized otoconia. This structure provides the mechanical stimulus necessary for hair cells of the vestibular maculae to respond to linear accelerations and gravity. In teleosts, Otolin is required for the proper anchoring of otolith crystals to the sensory maculae. Otoconia detachment and subsequent entrapment in the semicircular canals can result in benign paroxysmal positional vertigo (BPPV), a common form of vertigo for which the molecular basis is unknown. Several cDNAs encoding protein components of the mammalian otoconia and otoconial membrane have recently been identified, and mutations in these genes result in abnormal otoconia formation and balance deficits. Principal Findings Here we describe the cloning and characterization of mammalian Otolin, a protein constituent of otoconia and the otoconial membrane. Otolin is a secreted glycoprotein of ∼70 kDa, with a C-terminal globular domain that is homologous to the immune complement C1q, and contains extensive posttranslational modifications including hydroxylated prolines and glycosylated lysines. Like all C1q/TNF family members, Otolin multimerizes into higher order oligomeric complexes. The expression of otolin mRNA is restricted to the inner ear, and immunohistochemical analysis identified Otolin protein in support cells of the vestibular maculae and semi-circular canal cristae. Additionally, Otolin forms protein complexes with Cerebellin-1 and Otoconin-90, two protein constituents of the otoconia, when expressed in vitro. Otolin was also found in subsets of support cells and non-sensory cells of the cochlea, suggesting that Otolin is also a component of the tectorial membrane. Conclusion Given the importance of Otolin in lower organisms, the molecular cloning and biochemical characterization of the mammalian Otolin protein may lead to a better understanding of otoconial development and vestibular dysfunction. PMID:20856818

Pulse EPR distance measurements to study multimers and multimerisation

NASA Astrophysics Data System (ADS)

Ackermann, Katrin; Bode, Bela E.

2018-06-01

Pulse dipolar electron paramagnetic resonance (PD-EPR) has become a powerful tool for structural biology determining distances on the nanometre scale. Recent advances in hardware, methodology, and data analysis have widened the scope to complex biological systems. PD-EPR can be applied to systems containing lowly populated conformers or displaying large intrinsic flexibility, making them all but intractable for cryo-electron microscopy and crystallography. Membrane protein applications are of particular interest due to the intrinsic difficulties for obtaining high-resolution structures of all relevant conformations. Many drug targets involved in critical cell functions are multimeric channels or transporters. Here, common approaches for introducing spin labels for PD-EPR cause the presence of more than two electron spins per multimeric complex. This requires careful experimental design to overcome detrimental multi-spin effects and to secure sufficient distance resolution in presence of multiple distances. In addition to obtaining mere distances, PD-EPR can also provide information on multimerisation degrees allowing to study binding equilibria and to determine dissociation constants.

Interactions among tobacco sieve element occlusion (SEO) proteins.

PubMed

Jekat, Stephan B; Ernst, Antonia M; Zielonka, Sascia; Noll, Gundula A; Prüfer, Dirk

2012-12-01

Angiosperms transport their photoassimilates through sieve tubes, which comprise longitudinally-connected sieve elements. In dicots and also some monocots, the sieve elements contain parietal structural proteins known as phloem proteins or P-proteins. Following injury, P proteins disperse and accumulate as viscous plugs at the sieve plates to prevent the loss of valuable transport sugars. Tobacco (Nicotiana tabacum) P-proteins are multimeric complexes comprising subunits encoded by members of the SEO (sieve element occlusion) gene family. The existence of multiple subunits suggests that P-protein assembly involves interactions between SEO proteins, but this process is largely uncharacterized and it is unclear whether the different subunits perform unique roles or are redundant. We therefore extended our analysis of the tobacco P-proteins NtSEO1 and NtSEO2 to investigate potential interactions between them, and found that both proteins can form homomeric and heteromeric complexes in planta.

Facile and high-efficient immobilization of histidine-tagged multimeric protein G on magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Lee, Jiho; Chang, Jeong Ho

2014-12-01

This work reports the high-efficient and one-step immobilization of multimeric protein G on magnetic nanoparticles. The histidine-tagged (His-tag) recombinant multimeric protein G was overexpressed in Escherichia coli BL21 by the repeated linking of protein G monomers with a flexible linker. High-efficient immobilization on magnetic nanoparticles was demonstrated by two different preparation methods through the amino-silane and chloro-silane functionalization on silica-coated magnetic nanoparticles. Three kinds of multimeric protein G such as His-tag monomer, dimer, and trimer were tested for immobilization efficiency. For these tests, bicinchoninic acid (BCA) assay was employed to determine the amount of immobilized His-tag multimeric protein G. The result showed that the immobilization efficiency of the His-tag multimeric protein G of the monomer, dimer, and trimer was increased with the use of chloro-silane-functionalized magnetic nanoparticles in the range of 98% to 99%, rather than the use of amino-silane-functionalized magnetic nanoparticles in the range of 55% to 77%, respectively.

Phosphatidylinositol 4,5-Bisphosphate Clusters the Cell Adhesion Molecule CD44 and Assembles a Specific CD44-Ezrin Heterocomplex, as Revealed by Small Angle Neutron Scattering

DOE PAGES

Khajeh, Jahan Ali; Ju, Jeong Ho; Gupta, Yogesh K.; ...

2015-01-08

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin, which links the CD44 assembled receptor signaling complexes to the cytoskeletal actin and organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered and adopts an autoinhibited conformation, which prevents CD44ct from binding directly to activated Ezrin in solution. Binding to the signaling lipid phosphatidylinositol 4,5-biphosphlate (PIP2) disrupts autoinhibition in CD44ct, and activates CD44ct to associate with Ezrin.more » Further, using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific hetero-tetramer complex of CD44ct with Ezrin. This study reveals a novel autoregulation mechanism in the cytoplasmic tail of CD44 and the role of PIP2 in mediating the assembly of multimeric CD44ct-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of multimeric PIP2-CD44-Ezrin complexes.« less

Stochastic Protein Multimerization, Cooperativity and Fitness

NASA Astrophysics Data System (ADS)

Hagner, Kyle; Setayeshgar, Sima; Lynch, Michael

Many proteins assemble into multimeric structures that can vary greatly among phylogenetic lineages. As protein-protein interactions (PPI) require productive encounters among subunits, these structural variations are related in part to variation in cellular protein abundance. The protein abundance in turn depends on the intrinsic rates of production and decay of mRNA and protein molecules, as well as rates of cell growth and division. We present a stochastic model for prediction of the multimeric state of a protein as a function of these processes and the free energy associated with binding interfaces. We demonstrate favorable agreement between the model and a wide class of proteins using E. coli proteome data. As such, this platform, which links protein abundance, PPI and quaternary structure in growing and dividing cells can be extended to evolutionary models for the emergence and diversification of multimeric proteins. We investigate cooperativity - a ubiquitous functional property of multimeric proteins - as a possible selective force driving multimerization, demonstrating a reduction in the cost of protein production relative to the overall proteome energy budget that can be tied to fitness.

Formation of multimeric antibodies for self-delivery of active monomers.

PubMed

Dekel, Yaron; Machluf, Yossy; Gefen, Tal; Eidelshtein, Gennady; Kotlyar, Alexander; Bram, Yaron; Shahar, Ehud; Reslane, Farah; Aizenshtein, Elina; Pitcovski, Jacob

2017-11-01

Proteins and peptides have been used as drugs for almost a century. Technological advances in the past 30 years have enabled the production of pure, stable proteins in vast amounts. In contrast, administration of proteins based on their native active conformation (and thus necessitating the use of subcutaneous injections) has remained solely unchanged. The therapeutic anti-HER2 humanized monoclonal immunoglobulin (IgG) Trastuzumab (Herceptin) is a first line of the treatment for breast cancer. Chicken IgY is a commercially important polyclonal antibody (Ab). These Abs were examined for their ability to self-assemble and form ordered aggregates, by several biophysical methods. Atomic force microscopy analyses revealed the formation of multimeric nanostructures. The biological activity of multimeric IgG or IgY particles was retained and restored, in a dilution/time-dependent manner. IgG activity was confirmed by a binding assay using HER2 + human breast cancer cell line, SKBR3, while IgY activity was confirmed by ELISA assay using the VP2 antigen. Competition assay with native Herceptin antibodies demonstrated that the binding availability of the multimer formulation remained unaffected. Under long incubation periods, IgG multimers retained five times more activity than native IgG. In conclusion, the multimeric antibody formulations can serve as a storage depositories and sustained-release particles. These two important characteristics make this formulation promising for future novel administration protocols and altogether bring to light a different conceptual approach for the future use of therapeutic proteins as self-delivery entities rather than conjugated/encapsulated to other bio-compounds.

Evaluation of the effect of polymorphism on G-quadruplex-ligand interaction by means of spectroscopic and chromatographic techniques

NASA Astrophysics Data System (ADS)

Benito, S.; Ferrer, A.; Benabou, S.; Aviñó, A.; Eritja, R.; Gargallo, R.

2018-05-01

Guanine-rich sequences may fold into highly ordered structures known as G-quadruplexes. Apart from the monomeric G-quadruplex, these sequences may form multimeric structures that are not usually considered when studying interaction with ligands. This work studies the interaction of a ligand, crystal violet, with three guanine-rich DNA sequences with the capacity to form multimeric structures. These sequences correspond to short stretches found near the promoter regions of c-kit and SMARCA4 genes. Instrumental techniques (circular dichroism, molecular fluorescence, size-exclusion chromatography and electrospray ionization mass spectrometry) and multivariate data analysis were used for this purpose. The polymorphism of G-quadruplexes was characterized prior to the interaction studies. The ligand was shown to interact preferentially with the monomeric G-quadruplex; the binding stoichiometry was 1:1 and the binding constant was in the order of 105 M-1 for all three sequences. The results highlight the importance of DNA treatment prior to interaction studies.

Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

NASA Astrophysics Data System (ADS)

Parra-Belky, Karlett

2002-11-01

A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.

De Novo Design and Experimental Characterization of Ultrashort Self-Associating Peptides

PubMed Central

Xue, Bo; Robinson, Robert C.; Hauser, Charlotte A. E.; Floudas, Christodoulos A.

2014-01-01

Self-association is a common phenomenon in biology and one that can have positive and negative impacts, from the construction of the architectural cytoskeleton of cells to the formation of fibrils in amyloid diseases. Understanding the nature and mechanisms of self-association is important for modulating these systems and in creating biologically-inspired materials. Here, we present a two-stage de novo peptide design framework that can generate novel self-associating peptide systems. The first stage uses a simulated multimeric template structure as input into the optimization-based Sequence Selection to generate low potential energy sequences. The second stage is a computational validation procedure that calculates Fold Specificity and/or Approximate Association Affinity (K*association) based on metrics that we have devised for multimeric systems. This framework was applied to the design of self-associating tripeptides using the known self-associating tripeptide, Ac-IVD, as a structural template. Six computationally predicted tripeptides (Ac-LVE, Ac-YYD, Ac-LLE, Ac-YLD, Ac-MYD, Ac-VIE) were chosen for experimental validation in order to illustrate the self-association outcomes predicted by the three metrics. Self-association and electron microscopy studies revealed that Ac-LLE formed bead-like microstructures, Ac-LVE and Ac-YYD formed fibrillar aggregates, Ac-VIE and Ac-MYD formed hydrogels, and Ac-YLD crystallized under ambient conditions. An X-ray crystallographic study was carried out on a single crystal of Ac-YLD, which revealed that each molecule adopts a β-strand conformation that stack together to form parallel β-sheets. As an additional validation of the approach, the hydrogel-forming sequences of Ac-MYD and Ac-VIE were shuffled. The shuffled sequences were computationally predicted to have lower K*association values and were experimentally verified to not form hydrogels. This illustrates the robustness of the framework in predicting self-associating tripeptides. We expect that this enhanced multimeric de novo peptide design framework will find future application in creating novel self-associating peptides based on unnatural amino acids, and inhibitor peptides of detrimental self-aggregating biological proteins. PMID:25010703

Modeling the assembly order of multimeric heteroprotein complexes

PubMed Central

Esquivel-Rodriguez, Juan; Terashi, Genki; Christoffer, Charles; Shin, Woong-Hee

2018-01-01

Protein-protein interactions are the cornerstone of numerous biological processes. Although an increasing number of protein complex structures have been determined using experimental methods, relatively fewer studies have been performed to determine the assembly order of complexes. In addition to the insights into the molecular mechanisms of biological function provided by the structure of a complex, knowing the assembly order is important for understanding the process of complex formation. Assembly order is also practically useful for constructing subcomplexes as a step toward solving the entire complex experimentally, designing artificial protein complexes, and developing drugs that interrupt a critical step in the complex assembly. There are several experimental methods for determining the assembly order of complexes; however, these techniques are resource-intensive. Here, we present a computational method that predicts the assembly order of protein complexes by building the complex structure. The method, named Path-LzerD, uses a multimeric protein docking algorithm that assembles a protein complex structure from individual subunit structures and predicts assembly order by observing the simulated assembly process of the complex. Benchmarked on a dataset of complexes with experimental evidence of assembly order, Path-LZerD was successful in predicting the assembly pathway for the majority of the cases. Moreover, when compared with a simple approach that infers the assembly path from the buried surface area of subunits in the native complex, Path-LZerD has the strong advantage that it can be used for cases where the complex structure is not known. The path prediction accuracy decreased when starting from unbound monomers, particularly for larger complexes of five or more subunits, for which only a part of the assembly path was correctly identified. As the first method of its kind, Path-LZerD opens a new area of computational protein structure modeling and will be an indispensable approach for studying protein complexes. PMID:29329283

Modeling the assembly order of multimeric heteroprotein complexes.

PubMed

Peterson, Lenna X; Togawa, Yoichiro; Esquivel-Rodriguez, Juan; Terashi, Genki; Christoffer, Charles; Roy, Amitava; Shin, Woong-Hee; Kihara, Daisuke

2018-01-01

Protein-protein interactions are the cornerstone of numerous biological processes. Although an increasing number of protein complex structures have been determined using experimental methods, relatively fewer studies have been performed to determine the assembly order of complexes. In addition to the insights into the molecular mechanisms of biological function provided by the structure of a complex, knowing the assembly order is important for understanding the process of complex formation. Assembly order is also practically useful for constructing subcomplexes as a step toward solving the entire complex experimentally, designing artificial protein complexes, and developing drugs that interrupt a critical step in the complex assembly. There are several experimental methods for determining the assembly order of complexes; however, these techniques are resource-intensive. Here, we present a computational method that predicts the assembly order of protein complexes by building the complex structure. The method, named Path-LzerD, uses a multimeric protein docking algorithm that assembles a protein complex structure from individual subunit structures and predicts assembly order by observing the simulated assembly process of the complex. Benchmarked on a dataset of complexes with experimental evidence of assembly order, Path-LZerD was successful in predicting the assembly pathway for the majority of the cases. Moreover, when compared with a simple approach that infers the assembly path from the buried surface area of subunits in the native complex, Path-LZerD has the strong advantage that it can be used for cases where the complex structure is not known. The path prediction accuracy decreased when starting from unbound monomers, particularly for larger complexes of five or more subunits, for which only a part of the assembly path was correctly identified. As the first method of its kind, Path-LZerD opens a new area of computational protein structure modeling and will be an indispensable approach for studying protein complexes.

The assembly dynamics of the cytolytic pore toxin ClyA

PubMed Central

Benke, Stephan; Roderer, Daniel; Wunderlich, Bengt; Nettels, Daniel; Glockshuber, Rudi; Schuler, Benjamin

2015-01-01

Pore-forming toxins are protein assemblies used by many organisms to disrupt the membranes of target cells. They are expressed as soluble monomers that assemble spontaneously into multimeric pores. However, owing to their complexity, the assembly processes have not been resolved in detail for any pore-forming toxin. To determine the assembly mechanism for the ring-shaped, homododecameric pore of the bacterial cytolytic toxin ClyA, we collected a diverse set of kinetic data using single-molecule spectroscopy and complementary techniques on timescales from milliseconds to hours, and from picomolar to micromolar ClyA concentrations. The entire range of experimental results can be explained quantitatively by a surprisingly simple mechanism. First, addition of the detergent n-dodecyl-β-D-maltopyranoside to the soluble monomers triggers the formation of assembly-competent toxin subunits, accompanied by the transient formation of a molten-globule-like intermediate. Then, all sterically compatible oligomers contribute to assembly, which greatly enhances the efficiency of pore formation compared with simple monomer addition. PMID:25652783

Optimizing the relaxivity of GdIII complexes appended to InP/ZnS quantum dots by linker tuning.

PubMed

Stasiuk, Graeme J; Tamang, Sudarsan; Imbert, Daniel; Gateau, Christelle; Reiss, Peter; Fries, Pascal; Mazzanti, Marinella

2013-06-21

Three bimodal MRI/optical nanosized contrast agents with high per-nanoparticle relaxivity (up to 2523 mM(-1) s(-1) at 35 MHz and 932 mM(-1) s(-1) at 200 MHz) have been prepared connecting up to 115 tris-aqua Gd(III) complexes to fluorescent non-toxic InP/ZnS quantum dots. The structure of the linker has an important effect on the relaxivity of the final multimeric contrast agent.

Amyloidogenic Regions and Interaction Surfaces Overlap in Globular Proteins Related to Conformational Diseases

PubMed Central

Castillo, Virginia; Ventura, Salvador

2009-01-01

Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins. PMID:19696882

Multimerized CHR-derived peptides as HIV-1 fusion inhibitors.

PubMed

Nomura, Wataru; Hashimoto, Chie; Suzuki, Takaharu; Ohashi, Nami; Fujino, Masayuki; Murakami, Tsutomu; Yamamoto, Naoki; Tamamura, Hirokazu

2013-08-01

To date, several HIV-1 fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of an HIV-1 envelope protein gp41 have been discovered. We have shown that a synthetic peptide mimetic of a trimer form of the CHR-derived peptide C34 has potent inhibitory activity against the HIV-1 fusion mechanism, compared to a monomer C34 peptide. The present study revealed that a dimeric form of C34 is evidently structurally critical for fusion inhibitors, and that the activity of multimerized CHR-derived peptides in fusion inhibition is affected by the properties of the unit peptides C34, SC34EK, and T20. The fluorescence-based study suggested that the N36-interactive sites of the C34 trimer, including hydrophobic residues, are exposed outside the trimer and that trimerization of C34 caused a remarkable increase in fusion inhibitory activity. The present results could be useful in the design of fusion inhibitors against viral infections which proceed via membrane fusion with host cells. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Single-molecule force measurements of the polymerizing dimeric subunit of von Willebrand factor

NASA Astrophysics Data System (ADS)

Wijeratne, Sithara S.; Li, Jingqiang; Yeh, Hui-Chun; Nolasco, Leticia; Zhou, Zhou; Bergeron, Angela; Frey, Eric W.; Moake, Joel L.; Dong, Jing-fei; Kiang, Ching-Hwa

2016-01-01

Von Willebrand factor (VWF) multimers are large adhesive proteins that are essential to the initiation of hemostatic plugs at sites of vascular injury. The binding of VWF multimers to platelets, as well as VWF proteolysis, is regulated by shear stresses that alter VWF multimeric conformation. We used single molecule manipulation with atomic force microscopy (AFM) to investigate the effect of high fluid shear stress on soluble dimeric and multimeric forms of VWF. VWF dimers are the smallest unit that polymerizes to construct large VWF multimers. The resistance to mechanical unfolding with or without exposure to shear stress was used to evaluate VWF conformational forms. Our data indicate that, unlike recombinant VWF multimers (RVWF), recombinant dimeric VWF (RDVWF) unfolding force is not altered by high shear stress (100 dynes/cm2 for 3 min at 37°C ). We conclude that under the shear conditions used (100 dynes/cm2 for 3 min at 37°C ) , VWF dimers do not self-associate into a conformation analogous to that attained by sheared large VWF multimers.

Crystal structure of the Haemophilus influenzae Hap adhesin reveals an intercellular oligomerization mechanism for bacterial aggregation

PubMed Central

Meng, Guoyu; Spahich, Nicole; Kenjale, Roma; Waksman, Gabriel; St Geme, Joseph W

2011-01-01

Bacterial biofilms are complex microbial communities that are common in nature and are being recognized increasingly as an important determinant of bacterial virulence. However, the structural determinants of bacterial aggregation and eventual biofilm formation have been poorly defined. In Gram-negative bacteria, a major subgroup of extracellular proteins called self-associating autotransporters (SAATs) can mediate cell–cell adhesion and facilitate biofilm formation. In this study, we used the Haemophilus influenzae Hap autotransporter as a prototype SAAT to understand how bacteria associate with each other. The crystal structure of the H. influenzae HapS passenger domain (harbouring the SAAT domain) was determined to 2.2 Å by X-ray crystallography, revealing an unprecedented intercellular oligomerization mechanism for cell–cell interaction. The C-terminal SAAT domain folds into a triangular-prism-like structure that can mediate Hap–Hap dimerization and higher degrees of multimerization through its F1–F2 edge and F2 face. The intercellular multimerization can give rise to massive buried surfaces that are required for overcoming the repulsive force between cells, leading to bacterial cell–cell interaction and formation of complex microcolonies. PMID:21841773

Collision induced unfolding of isolated proteins in the gas phase: past, present, and future.

PubMed

Dixit, Sugyan M; Polasky, Daniel A; Ruotolo, Brandon T

2018-02-01

Rapidly characterizing the three-dimensional structures of proteins and the multimeric machines they form remains one of the great challenges facing modern biological and medical sciences. Ion mobility-mass spectrometry based techniques are playing an expanding role in characterizing these functional complexes, especially in drug discovery and development workflows. Despite this expansion, ion mobility-mass spectrometry faces many challenges, especially in the context of detecting small differences in protein tertiary structure that bear functional consequences. Collision induced unfolding is an ion mobility-mass spectrometry method that enables the rapid differentiation of subtly-different protein isoforms based on their unfolding patterns and stabilities. In this review, we summarize the modern implementation of such gas-phase unfolding experiments and provide an overview of recent developments in both methods and applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Conformational changes of the N-terminal part of Mason-Pfizer monkey virus p12 protein during multimerization

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

Knejzlik, Zdenek; Ulbrich, Pavel; Strohalm, Martin

2009-10-10

The Mason-Pfizer monkey virus is a prototype Betaretrovirus with the defining characteristic that it assembles spherical immature particles from Gag-related polyprotein precursors within the cytoplasm of the infected cell. It was shown previously that the N-terminal part of the Gag p12 domain (wt-Np12) is required for efficient assembly. However, the precise role for p12 in mediating Gag-Gag interaction is still poorly understood. In this study we employed detailed circular dichroism spectroscopy, electron microscopy and ultracentrifugation analyses of recombinant wt-Np12 prepared by in vitro transcription and translation. The wt-Np12 domain fragment forms fibrillar structures in a concentration-dependent manner. Assembly into fibersmore » is linked to a conformational transition from unfolded or another non-periodical state to alpha-helix during multimerization.« less

Structural Evidence for the Tetrameric Assembly of Chemokine CCL11 and the Glycosaminoglycan Arixtra™

PubMed Central

Dykstra, Andrew B.; Sweeney, Matt D.; Leary, Julie A.

2013-01-01

Understanding chemokine interactions with glycosaminoglycans (GAG) is critical as these interactions have been linked to a number of inflammatory medical conditions, such as arthritis and asthma. To better characterize in vivo protein function, comprehensive knowledge of multimeric species, formed by chemokines under native conditions, is necessary. Herein is the first report of a tetrameric assembly of the human chemokine CCL11, which was shown bound to the GAG Arixtra™. Isothermal titration calorimetry data indicated that CCL11 interacts with Arixtra, and ion mobility mass spectrometry (IM-MS) was used to identify ions corresponding to the CCL11 tetrameric species bound to Arixtra. Collisional cross sections (CCS) of the CCL11 tetramer-Arixtra noncovalent complex were compared to theoretical CCS values calculated using a preliminary structure of the complex deduced using X-ray crystallography. Experimental CCS values were in agreement with theoretical values, strengthening the IM-MS evidence for the formation of the noncovalent complex. Tandem mass spectrometry data of the complex indicated that the tetramer-GAG complex dissociates into a monomer and a trimer-GAG species, suggesting that two CC-like dimers are bridged by Arixtra. As development of chemokine inhibitors is of utmost importance to treatment of medical inflammatory conditions, these results provide vital insights into chemokine-GAG interactions. PMID:24970196

Structural Evidence for the Tetrameric Assembly of Chemokine CCL11 and the Glycosaminoglycan Arixtra™.

PubMed

Dykstra, Andrew B; Sweeney, Matt D; Leary, Julie A

2013-11-06

Understanding chemokine interactions with glycosaminoglycans (GAG) is critical as these interactions have been linked to a number of inflammatory medical conditions, such as arthritis and asthma. To better characterize in vivo protein function, comprehensive knowledge of multimeric species, formed by chemokines under native conditions, is necessary. Herein is the first report of a tetrameric assembly of the human chemokine CCL11, which was shown bound to the GAG Arixtra™. Isothermal titration calorimetry data indicated that CCL11 interacts with Arixtra, and ion mobility mass spectrometry (IM-MS) was used to identify ions corresponding to the CCL11 tetrameric species bound to Arixtra. Collisional cross sections (CCS) of the CCL11 tetramer-Arixtra noncovalent complex were compared to theoretical CCS values calculated using a preliminary structure of the complex deduced using X-ray crystallography. Experimental CCS values were in agreement with theoretical values, strengthening the IM-MS evidence for the formation of the noncovalent complex. Tandem mass spectrometry data of the complex indicated that the tetramer-GAG complex dissociates into a monomer and a trimer-GAG species, suggesting that two CC-like dimers are bridged by Arixtra. As development of chemokine inhibitors is of utmost importance to treatment of medical inflammatory conditions, these results provide vital insights into chemokine-GAG interactions.

Regulation of SMN Protein Stability▿ †

PubMed Central

Burnett, Barrington G.; Muñoz, Eric; Tandon, Animesh; Kwon, Deborah Y.; Sumner, Charlotte J.; Fischbeck, Kenneth H.

2009-01-01

Spinal muscular atrophy (SMA) is caused by mutations of the survival of motor neuron (SMN1) gene and deficiency of full-length SMN protein (FL-SMN). All SMA patients retain one or more copies of the SMN2 gene, but the principal protein product of SMN2 lacks exon 7 (SMNΔ7) and is unable to compensate for a deficiency of FL-SMN. SMN is known to oligomerize and form a multimeric protein complex; however, the mechanisms regulating stability and degradation of FL-SMN and SMNΔ7 proteins have been largely unexplored. Using pulse-chase analysis, we characterized SMN protein turnover and confirmed that SMN was ubiquitinated and degraded by the ubiquitin proteasome system (UPS). The SMNΔ7 protein had a twofold shorter half-life than FL-SMN in cells despite similar intrinsic rates of turnover by the UPS in a cell-free assay. Mutations that inhibited SMN oligomerization and complex formation reduced the FL-SMN half-life. Furthermore, recruitment of SMN into large macromolecular complexes as well as increased association with several Gemin proteins was regulated in part by protein kinase A. Together, our data indicate that SMN protein stability is modulated by complex formation. Promotion of the SMN complex formation may be an important novel therapeutic strategy for SMA. PMID:19103745

Effects of introducing a single charged residue into the phenylalanine clamp of multimeric anthrax protective antigen.

PubMed

Janowiak, Blythe E; Fischer, Audrey; Collier, R John

2010-03-12

Multimeric pores formed in the endosomal membrane by the Protective Antigen moiety of anthrax toxin translocate the enzymatic moieties of the toxin to the cytosolic compartment of mammalian cells. There is evidence that the side chains of the Phe(427) residues come into close proximity with one another in the lumen of the pore and form a structure, termed the Phe clamp, that catalyzes the translocation process. In this report we describe the effects of replacing Phe(427) in a single subunit of the predominantly heptameric pore with a basic or an acidic amino acid. Incorporating any charged residue at this position inhibited cytotoxicity >or=1,000-fold in our standard assay and caused strong inhibition of translocation in a planar phospholipid bilayer system. His and Glu were the most strongly inhibitory residues, ablating both cytotoxicity and translocation. Basic residues at position 427 prevented the Phe clamp from interacting with a translocation substrate to form a seal against the passage of ions and accelerated dissociation of the substrate from the pore. Acidic residues, in contrast, allowed the seal to form and the substrate to remain firmly bound, but blocked its passage, perhaps via electrostatic interactions with the positively charged N-terminal segment. Our findings are discussed in relation to the role of the Phe clamp in a Brownian ratchet model of translocation.

Distinct subcellular trafficking resulting from monomeric vs multimeric targeting to endothelial ICAM-1: implications for drug delivery.

PubMed

Ghaffarian, Rasa; Muro, Silvia

2014-12-01

Ligand-targeted, receptor-mediated endocytosis is commonly exploited for intracellular drug delivery. However, cells-surface receptors may follow distinct endocytic fates when bound by monomeric vs multimeric ligands. Our purpose was to study this paradigm using ICAM-1, an endothelial receptor involved in inflammation, to better understand its regulation and potential for drug delivery. Our procedure involved fluorescence microscopy of human endothelial cells to determine the endocytic behavior of unbound ICAM-1 vs ICAM-1 bound by model ligands: monomeric (anti-ICAM) vs multimeric (anti-ICAM biotin-streptavidin conjugates or anti-ICAM coated onto 100 nm nanocarriers). Our findings suggest that both monomeric and multimeric ligands undergo a similar endocytic pathway sensitive to amiloride (∼50% inhibition), but not inhibitors of clathrin-pits or caveoli. After 30 min, ∼60-70% of both ligands colocalized with Rab11a-compartments. By 3-5 h, ∼65-80% of multimeric anti-ICAM colocalized with perinuclear lysosomes with ∼60-80% degradation, while 70% of monomeric anti-ICAM remained associated with Rab11a at the cell periphery and recycled to and from the cell-surface with minimal (<10%) lysosomal colocalization and minimal (≤15%) degradation. In the absence of ligands, ICAM-1 also underwent amiloride-sensitive endocytosis with peripheral distribution, suggesting that monomeric (not multimeric) anti-ICAM follows the route of this receptor. In conclusion, ICAM-1 can mediate different intracellular itineraries, revealing new insight into this biological pathway and alternative avenues for drug delivery.

Highly efficient antibody immobilization with multimeric protein Gs coupled magnetic silica nanoparticles

NASA Astrophysics Data System (ADS)

Lee, J. H.; Choi, H. K.; Chang, J. H.

2011-10-01

This work reports the immobilization of monomeric, dimeric and trimer protein Gs onto silica magnetic nanoparticles for self-oriented antibody immobilization. To achieve this, we initially prepared the silica-coated magnetic nanoparticle having about 170 nm diameters. The surface of the silica coated magnetic nanoparticles was modified with 3- aminopropyl-trimethoxysilane (APTMS) to chemically link to multimeric protein Gs. The conjugation of amino groups on the SiO2-MNPs to cysteine tagged in multimeric protein Gs was performed using a sulfo-SMCC coupling procedure. The binding efficiencies of monomer, dimer and trimer were 77 %, 67 % and 55 % respectively. However, the efficiencies of antibody immobilization were 70 %, 83 % and 95 % for monomeric, dimeric and trimeric protein G, respectively. To prove the enhancement of accessibility by using multimeric protein G, FITC labeled goat-anti-mouse IgG was treated to mouse IgG immobilized magnetic silica nanoparticles through multimeric protein G. FITC labeled goat anti-mouse IgGs were more easily bound to mouse IgG immobilized by trimeric protein G than others. Finally protein G bound silica magnetic nanoparticles were utilized to develop highly sensitive immunoassay to detect hepatitis B antigen.

A Rolling Circle Replication Mechanism Produces Multimeric Lariats of Mitochondrial DNA in Caenorhabditis elegans

PubMed Central

Lewis, Samantha C.; Joers, Priit; Willcox, Smaranda; Griffith, Jack D.; Jacobs, Howard T.; Hyman, Bradley C.

2015-01-01

Mitochondrial DNA (mtDNA) encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s) of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s) of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans. PMID:25693201

Characterization of auxin-binding proteins from zucchini plasma membrane

NASA Technical Reports Server (NTRS)

Hicks, G. R.; Rice, M. S.; Lomax, T. L.

1993-01-01

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may possess transporter or channel function.

Characterization of auxin-binding proteins from zucchini plasma membrane.

PubMed

Hicks, G R; Rice, M S; Lomax, T L

1993-01-01

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may possess transporter or channel function.

Induced Fit in Protein Multimerization: The HFBI Case

PubMed Central

Riccardi, Laura

2016-01-01

Hydrophobins, produced by filamentous fungi, are small amphipathic proteins whose biological functions rely on their unique surface-activity properties. Understanding the mechanistic details of the multimerization process is of primary importance to clarify the interfacial activity of hydrophobins. We used free energy calculations to study the role of a flexible β-hairpin in the multimerization process in hydrophobin II from Trichoderma reesei (HFBI). We characterized how the displacement of this β-hairpin controls the stability of the monomers/dimers/tetramers in solution. The regulation of the oligomerization equilibrium of HFBI will necessarily affect its interfacial properties, fundamental for its biological function and for technological applications. Moreover, we propose possible routes for the multimerization process of HFBI in solution. This is the first case where a mechanism by which a flexible loop flanking a rigid patch controls the protein-protein binding equilibrium, already known for proteins with charged binding hot-spots, is described within a hydrophobic patch. PMID:27832079

Oligomeric state of purified transient receptor potential melastatin-1 (TRPM1), a protein essential for dim light vision.

PubMed

Agosto, Melina A; Zhang, Zhixian; He, Feng; Anastassov, Ivan A; Wright, Sara J; McGehee, Jennifer; Wensel, Theodore G

2014-09-26

Transient receptor potential melastatin-1 (TRPM1) is essential for the light-induced depolarization of retinal ON bipolar cells. TRPM1 likely forms a multimeric channel complex, although almost nothing is known about the structure or subunit composition of channels formed by TRPM1 or any of its close relatives. Recombinant TRPM1 was robustly expressed in insect cells, but only a small fraction was localized to the plasma membrane. Similar intracellular localization was observed when TRPM1 was heterologously expressed in mammalian cells. TRPM1 was affinity-purified from Sf9 cells and complexed with amphipol, followed by detergent removal. In blue native gels and size exclusion chromatography, TRPM1 migrated with a mobility consistent with detergent- or amphipol-bound dimers. Cross-linking experiments were also consistent with a dimeric subunit stoichiometry, and cryoelectron microscopy and single particle analysis without symmetry imposition yielded a model with approximate 2-fold symmetrical features. Finally, electron microscopy of TRPM1-antibody complexes revealed a large particle that can accommodate TRPM1 and two antibody molecules. Taken together, these data indicate that purified TRPM1 is mostly dimeric. The three-dimensional structure of TRPM1 dimers is characterized by a small putative transmembrane domain and a larger domain with a hollow cavity. Blue native gels of solubilized mouse retina indicate that TRPM1 is present in two distinct complexes: one similar in size to the recombinant protein and one much larger. Because dimers are likely not functional ion channels, these results suggest that additional partner subunits participate in forming the transduction channel required for dim light vision and the ON pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular interactions of orthologues of floral homeotic proteins from the gymnosperm Gnetum gnemon provide a clue to the evolutionary origin of 'floral quartets'.

PubMed

Wang, Yong-Qiang; Melzer, Rainer; Theissen, Günter

2010-10-01

Several lines of evidence suggest that the identity of floral organs in angiosperms is specified by multimeric transcription factor complexes composed of MADS-domain proteins. These bind to specific cis-regulatory elements ('CArG-boxes') of their target genes involving DNA-loop formation, thus constituting 'floral quartets'. Gymnosperms, angiosperms' closest relatives, contain orthologues of floral homeotic genes, but when and how the interactions constituting floral quartets were established during evolution has remained unknown. We have comprehensively studied the dimerization and DNA-binding of several classes of MADS-domain proteins from the gymnosperm Gnetum gnemon. Determination of protein-protein and protein-DNA interactions by yeast two-hybrid, in vitro pull-down and electrophoretic mobility shift assays revealed complex patterns of homo- and heterodimerization among orthologues of floral homeotic class B, class C and class E proteins and B(sister) proteins. Using DNase I footprint assays we demonstrate that both orthologues of class B with C proteins, and orthologues of class C proteins alone, but not orthologues of class B proteins alone can loop DNA in floral quartet-like complexes. This is in contrast to class B and class C proteins from angiosperms, which require other factors such as class E floral homeotic proteins to 'glue' them together in multimeric complexes. Our findings suggest that the evolutionary origin of floral quartet formation is based on the interaction of different DNA-bound homodimers, does not depend on class E proteins, and predates the origin of angiosperms. © 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.

Structure of the thermophilic l-Arabinose isomerase from Geobacillus kaustophilus reveals metal-mediated intersubunit interactions for activity and thermostability.

PubMed

Choi, Jin Myung; Lee, Yong-Jik; Cao, Thinh-Phat; Shin, Sun-Mi; Park, Min-Kyu; Lee, Han-Seung; di Luccio, Eric; Kim, Seong-Bo; Lee, Sang-Jae; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong-Woo

2016-04-15

Thermophilic l-arabinose isomerase (AI), which catalyzes the interconversion of l-arabinose and l-ribulose, can be used to produce d-tagatose, a sugar substitute, from d-galactose. Unlike mesophilic AIs, thermophilic AIs are highly dependent on divalent metal ions for their catalytic activity and thermostability at elevated temperatures. However, the molecular basis underlying the substrate preferences and metal requirements of multimeric AIs remains unclear. Here we report the first crystal structure of the apo and holo forms of thermophilic Geobacillus kaustophilus AI (GKAI) in hexamer form. The structures, including those of GKAI in complex with l-arabitol, and biochemical analyses revealed not only how the substrate-binding site of GKAI is formed through displacement of residues at the intersubunit interface when it is bound to Mn(2+), but also revealed the water-mediated H-bonding networks that contribute to the structural integrity of GKAI during catalysis. These observations suggest metal-mediated isomerization reactions brought about by intersubunit interactions at elevated temperatures are responsible for the distinct active site features that promote the substrate specificity and thermostability of thermophilic AIs. Copyright © 2016 Elsevier Inc. All rights reserved.

Structure-function analyses unravel distinct effects of allosteric inhibitors of HIV-1 integrase on viral maturation and integration.

PubMed

Bonnard, Damien; Le Rouzic, Erwann; Eiler, Sylvia; Amadori, Céline; Orlov, Igor; Bruneau, Jean-Michel; Brias, Julie; Barbion, Julien; Chevreuil, Francis; Spehner, Danièle; Chasset, Sophie; Ledoussal, Benoit; Moreau, François; Saïb, Ali; Klaholz, Bruno P; Emiliani, Stéphane; Ruff, Marc; Zamborlini, Alessia; Benarous, Richard

2018-04-20

Recently, a new class of HIV-1 integrase (IN) inhibitors with a dual mode of action, called IN-LEDGF/p75 allosteric inhibitors (INLAIs), was described. Designed to interfere with the IN-LEDGF/p75 interaction during viral integration, unexpectedly, their major impact was on virus maturation. This activity has been linked to induction of aberrant IN multimerization, whereas inhibition of the IN-LEDGF/p75 interaction accounts for weaker antiretroviral effect at integration. Because these dual activities result from INLAI binding to IN at a single binding site, we expected that these activities co-evolved together, driven by the affinity for IN. Using an original INLAI, MUT-A, and its activity on an Ala-125 (A125) IN variant, we found that these two activities on A125-IN can be fully dissociated: MUT-A-induced IN multimerization and the formation of eccentric condensates in viral particles, which are responsible for inhibition of virus maturation, were lost, whereas inhibition of the IN-LEDGF/p75 interaction and consequently integration was fully retained. Hence, the mere binding of INLAI to A125 IN is insufficient to promote the conformational changes of IN required for aberrant multimerization. By analyzing the X-ray structures of MUT-A bound to the IN catalytic core domain (CCD) with or without the Ala-125 polymorphism, we discovered that the loss of IN multimerization is due to stabilization of the A125-IN variant CCD dimer, highlighting the importance of the CCD dimerization energy for IN multimerization. Our study reveals that affinity for the LEDGF/p75-binding pocket is not sufficient to induce INLAI-dependent IN multimerization and the associated inhibition of viral maturation. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular basis for multimerization in the activation of the epidermal growth factor receptor

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

Huang, Yongjian; Bharill, Shashank; Karandur, Deepti

The epidermal growth factor receptor (EGFR) is activated by dimerization, but activation also generates higher-order multimers, whose nature and function are poorly understood. We have characterized ligand-induced dimerization and multimerization of EGFR using single-molecule analysis, and show that multimerization can be blocked by mutations in a specific region of Domain IV of the extracellular module. These mutations reduce autophosphorylation of the C-terminal tail of EGFR and attenuate phosphorylation of phosphatidyl inositol 3-kinase, which is recruited by EGFR. The catalytic activity of EGFR is switched on through allosteric activation of one kinase domain by another, and we show that if thismore » is restricted to dimers, then sites in the tail that are proximal to the kinase domain are phosphorylated in only one subunit. We propose a structural model for EGFR multimerization through self-association of ligand-bound dimers, in which the majority of kinase domains are activated cooperatively, thereby boosting tail phosphorylation.« less

Molecular basis for multimerization in the activation of the epidermal growth factor receptor

DOE PAGES

Huang, Yongjian; Bharill, Shashank; Karandur, Deepti; ...

2016-03-28

The epidermal growth factor receptor (EGFR) is activated by dimerization, but activation also generates higher-order multimers, whose nature and function are poorly understood. We have characterized ligand-induced dimerization and multimerization of EGFR using single-molecule analysis, and show that multimerization can be blocked by mutations in a specific region of Domain IV of the extracellular module. These mutations reduce autophosphorylation of the C-terminal tail of EGFR and attenuate phosphorylation of phosphatidyl inositol 3-kinase, which is recruited by EGFR. The catalytic activity of EGFR is switched on through allosteric activation of one kinase domain by another, and we show that if thismore » is restricted to dimers, then sites in the tail that are proximal to the kinase domain are phosphorylated in only one subunit. We propose a structural model for EGFR multimerization through self-association of ligand-bound dimers, in which the majority of kinase domains are activated cooperatively, thereby boosting tail phosphorylation.« less

Rapid acquisition of beta-sheet structure in the prion protein prior to multimer formation.

PubMed

Post, K; Pitschke, M; Schäfer, O; Wille, H; Appel, T R; Kirsch, D; Mehlhorn, I; Serban, H; Prusiner, S B; Riesner, D

1998-11-01

The N-terminally truncated form of the prion protein, PrP 27-30, and the corresponding recombinant protein, rPrP, were solubilized in 0.2% SDS, and the transitions induced by changing the conditions from 0.2% SDS to physiological conditions, i.e. removing SDS, were characterized with respect to solubility, resistance to proteolysis, secondary structure and multimerization. Circular dichroism, electron microscopy and fluorescence correlation spectroscopy were used to study the structural transitions of PrP. Within one minute the alpha-helical structure of PrP was transformed into one that was enriched in beta-sheets and consisted mainly of dimers. Larger oligomers were found after 20 minutes and larger multimers exhibiting resistance to proteolysis were found after several hours. It was concluded that the monomeric alpha-helical conformation was stable in SDS or when attached to the membrane; however, the state of lowest free energy in aqueous solution at neutral pH seems to be the multimeric, beta-sheet enriched conformation.

PML/RARA oxidation and arsenic binding initiate the antileukemia response of As2O3.

PubMed

Jeanne, Marion; Lallemand-Breitenbach, Valérie; Ferhi, Omar; Koken, Marcel; Le Bras, Morgane; Duffort, Stéphanie; Peres, Laurent; Berthier, Caroline; Soilihi, Hassane; Raught, Brian; de Thé, Hugues

2010-07-13

As(2)O(3) cures acute promyelocytic leukemia (APL) by initiating PML/RARA oncoprotein degradation, through sumoylation of its PML moiety. However, how As(2)O(3) initiates PML sumoylation has remained largely unexplained. As(2)O(3) binds vicinal cysteines and increases reactive oxygen species (ROS) production. We demonstrate that upon As(2)O(3) exposure, PML undergoes ROS-initiated intermolecular disulfide formation and binds arsenic directly. Disulfide-linked PML or PML/RARA multimers form nuclear matrix-associated nuclear bodies (NBs), become sumoylated and are degraded. Hematopoietic progenitors transformed by an As(2)O(3)-binding PML/RARA mutant exhibit defective As(2)O(3) response. Conversely, nonarsenical oxidants elicit PML/RARA multimerization, NB-association, degradation, and leukemia response in vivo, but do not affect PLZF/RARA-driven APLs. Thus, PML oxidation regulates NB-biogenesis, while oxidation-enforced PML/RARA multimerization and direct arsenic-binding cooperate to enforce APL's exquisite As(2)O(3) sensitivity. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Von Willebrand's disease: case report and review of literature.

PubMed

Echahdi, Hanae; El Hasbaoui, Brahim; El Khorassani, Mohamed; Agadr, Aomar; Khattab, Mohamed

2017-01-01

Von Willebrand Disease (VWD) is the most common human inherited bleeding disorder due to a defect of Von Willebrand Factor (VWF), which a glycoprotein crucial for platelet adhesion to the subendothelium after vascular injury. VWD include quantitative defects of VWF, either partial (type 1 with VWF levels < 50 IU/dL) or virtually total (type 3 with undetectable VWF levels) and also qualitative defects of VWF (type 2 variants with discrepant antigenic and functional VWF levels). The most bleeding forms of VWD usually do not concern type 1 patients with the mildest VWF defects (VWF levels between 30 and 50IU/dL). Von willebrand factor is a complex multimeric protein with two functions: it forms a bridge between the platelets and areas of vascular damage and it binds to and stabilizes factor VIII, which is necessary for the clotting cascade. By taking a clinical history of bleeding (mucocutaneous bleeding symptoms suggestive of a primary haemostatic disorder, a quantitative or qualitative abnormality of VWF is possible) it is important to think about VWD and to make the appropriate diagnosis. If the VWD is suspected diagnostic tests should include an activated partial thromboplastin time, bleeding time, factor VIII: C Ristocetin cofactor and vWF antigen. Additional testing of ristocetin induced plattlet adhesion (RIPA) the multimeric structure and collagen binding test and genanalysis allow diagnosing the different types of von. Willebrand Disease. The treatment of choice in mild forms is the synthetic agent desmopressin. In patients with severe type 1, type 2B, 2N and type 3 or in people who do not response to desmopressin, the appropriate treatment is a factor VIII concentrate that is rich of VWF. We report a case of infant in 27-month-old boy who had been referred due to haemorrhagic shock. His birth histories, his familie's social history and developmental milestones were unremarkable. He was born at full term with no antenatal or perinatal complications. Prior to the symptoms, the child was on a normal diet and was thriving appropriately. The child presented one days before his admission trauma to the inner face of the lower lip that caused an external acute bleeding loss. The laboratory data showed unfortunately, the most severe form of Von Willebrand's Disease; Type 3. The management was based on erythrocyte and fresh-frozen plasma (FFP) transfusions with administration of factor VII with good evolution.

Cytokine-like factor-1, a novel soluble protein, shares homology with members of the cytokine type I receptor family.

PubMed

Elson, G C; Graber, P; Losberger, C; Herren, S; Gretener, D; Menoud, L N; Wells, T N; Kosco-Vilbois, M H; Gauchat, J F

1998-08-01

In this report we describe the identification, cloning, and expression pattern of human cytokine-like factor 1 (hCLF-1) and the identification and cloning of its murine homologue. They were identified from expressed sequence tags using amino acid sequences from conserved regions of the cytokine type I receptor family. Human CLF-1 and murine CLF-1 shared 96% amino acid identity and significant homology with many cytokine type I receptors. CLF-1 is a secreted protein, suggesting that it is either a soluble subunit within a cytokine receptor complex, like the soluble form of the IL-6R alpha-chain, or a subunit of a multimeric cytokine, e.g., IL-12 p40. The highest levels of hCLF-1 mRNA were observed in lymph node, spleen, thymus, appendix, placenta, stomach, bone marrow, and fetal lung, with constitutive expression of CLF-1 mRNA detected in a human kidney fibroblastic cell line. In fibroblast primary cell cultures, CLF-1 mRNA was up-regulated by TNF-alpha, IL-6, and IFN-gamma. Western blot analysis of recombinant forms of hCLF-1 showed that the protein has the tendency to form covalently linked di- and tetramers. These results suggest that CLF-1 is a novel soluble cytokine receptor subunit or part of a novel cytokine complex, possibly playing a regulatory role in the immune system and during fetal development.

3D Complex: A Structural Classification of Protein Complexes

PubMed Central

Levy, Emmanuel D; Pereira-Leal, Jose B; Chothia, Cyrus; Teichmann, Sarah A

2006-01-01

Most of the proteins in a cell assemble into complexes to carry out their function. It is therefore crucial to understand the physicochemical properties as well as the evolution of interactions between proteins. The Protein Data Bank represents an important source of information for such studies, because more than half of the structures are homo- or heteromeric protein complexes. Here we propose the first hierarchical classification of whole protein complexes of known 3-D structure, based on representing their fundamental structural features as a graph. This classification provides the first overview of all the complexes in the Protein Data Bank and allows nonredundant sets to be derived at different levels of detail. This reveals that between one-half and two-thirds of known structures are multimeric, depending on the level of redundancy accepted. We also analyse the structures in terms of the topological arrangement of their subunits and find that they form a small number of arrangements compared with all theoretically possible ones. This is because most complexes contain four subunits or less, and the large majority are homomeric. In addition, there is a strong tendency for symmetry in complexes, even for heteromeric complexes. Finally, through comparison of Biological Units in the Protein Data Bank with the Protein Quaternary Structure database, we identified many possible errors in quaternary structure assignments. Our classification, available as a database and Web server at http://www.3Dcomplex.org, will be a starting point for future work aimed at understanding the structure and evolution of protein complexes. PMID:17112313

The coactivator CBP stimulates human T-cell lymphotrophic virus type I Tax transactivation in vitro.

PubMed

Kashanchi, F; Duvall, J F; Kwok, R P; Lundblad, J R; Goodman, R H; Brady, J N

1998-12-18

Tax interacts with the cellular cyclic AMP-responsive element binding protein (CREB) and facilitates the binding of the coactivator CREB binding protein (CBP), forming a multimeric complex on the cyclic AMP-responsive element (CRE)-like sites in the human T-cell lymphotrophic virus type I (HTLV-I) promoter. The trimeric complex is believed to recruit additional regulatory proteins to the HTLV-I long terminal repeat, but there has been no direct evidence that CBP is required for Tax-mediated transactivation. We present evidence that Tax and CBP activate transcription from the HTLV-I 21 base pair repeats on naked DNA templates. Transcriptional activation of the HTLV-I sequences required both Tax and CBP and could be mediated by either the N-terminal activation domain of CBP or the full-length protein. Fluorescence polarization binding assays indicated that CBP does not markedly enhance the affinity of Tax for the trimeric complex. Transcription analyses suggest that CBP activates Tax-dependent transcription by promoting transcriptional initiation and reinitiation. The ability of CBP to activate the HTLV-I promoter does not involve the stabilization of Tax binding, but rather depends upon gene activation properties of the co-activator that function in the context of a naked DNA template.

Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities.

PubMed

Wang, Guoping; Ding, Xiong; Hu, Jiumei; Wu, Wenshuai; Sun, Jingjing; Mu, Ying

2017-10-24

Existing isothermal nucleic acid amplification (INAA) relying on the strand displacement activity of DNA polymerase usually requires at least two primers. However, in this paper, we report an unusual isothermal multimerization and amplification (UIMA) which only needs one primer and is efficiently initiated by the strand-displacing DNA polymerases with reverse transcription activities. On electrophoresis, the products of UIMA present a cascade-shape band and they are confirmed to be multimeric DNAs with repeated target sequences. In contrast to current methods, UIMA is simple to product multimeric DNA, due to the independent of multiple primers and rolling circle structures. Through assaying the synthesized single-stranded DNA targets, UIMA performs high sensitivity and specificity, as well as the universality. In addition, a plausible mechanism of UIMA is proposed, involving short DNA bending, mismatch extension, and template slippage. UIMA is a good explanation for why nonspecific amplification easily happens in existing INAAs. As the simplest INAA till now, UIMA provides a new insight for deeply understanding INAA and opens a new avenue for thoroughly addressing nonspecific amplification.

The Tumor Suppressor DiRas3 Forms a Complex with H-Ras and C-RAF Proteins and Regulates Localization, Dimerization, and Kinase Activity of C-RAF*

PubMed Central

Baljuls, Angela; Beck, Matthias; Oenel, Ayla; Robubi, Armin; Kroschewski, Ruth; Hekman, Mirko; Rudel, Thomas; Rapp, Ulf R.

2012-01-01

The maternally imprinted Ras-related tumor suppressor gene DiRas3 is lost or down-regulated in more than 60% of ovarian and breast cancers. The anti-tumorigenic effect of DiRas3 is achieved through several mechanisms, including inhibition of cell proliferation, motility, and invasion, as well as induction of apoptosis and autophagy. Re-expression of DiRas3 in cancer cells interferes with the signaling through Ras/MAPK and PI3K. Despite intensive research, the mode of interference of DiRas3 with the Ras/RAF/MEK/ERK signal transduction is still a matter of speculation. In this study, we show that DiRas3 associates with the H-Ras oncogene and that activation of H-Ras enforces this interaction. Furthermore, while associated with DiRas3, H-Ras is able to bind to its effector protein C-RAF. The resulting multimeric complex consisting of DiRas3, C-RAF, and active H-Ras is more stable than the two protein complexes H-Ras·C-RAF or H-Ras·DiRas3, respectively. The consequence of this complex formation is a DiRas3-mediated recruitment and anchorage of C-RAF to components of the membrane skeleton, suppression of C-RAF/B-RAF heterodimerization, and inhibition of C-RAF kinase activity. PMID:22605333

Interaction of glutaric aciduria type 1-related glutaryl-CoA dehydrogenase with mitochondrial matrix proteins.

PubMed

Schmiesing, Jessica; Schlüter, Hartmut; Ullrich, Kurt; Braulke, Thomas; Mühlhausen, Chris

2014-01-01

Glutaric aciduria type 1 (GA1) is an inherited neurometabolic disorder caused by mutations in the GCDH gene encoding glutaryl-CoA dehydrogenase (GCDH), which forms homo- and heteromeric complexes in the mitochondrial matrix. GA1 patients are prone to the development of encephalopathic crises which lead to an irreversible disabling dystonic movement disorder. The clinical and biochemical manifestations of GA1 vary considerably and lack correlations to the genotype. Using an affinity chromatography approach we report here for the first time on the identification of mitochondrial proteins interacting directly with GCDH. Among others, dihydrolipoamide S-succinyltransferase (DLST) involved in the formation of glutaryl-CoA, and the β-subunit of the electron transfer flavoprotein (ETFB) serving as electron acceptor, were identified as GCDH binding partners. We have adapted the yellow fluorescent protein-based fragment complementation assay and visualized the oligomerization of GCDH as well as its direct interaction with DLST and ETFB in mitochondria of living cells. These data suggest that GCDH is a constituent of multimeric mitochondrial dehydrogenase complexes, and the characterization of their interrelated functions may provide new insights into the regulation of lysine oxidation and the pathophysiology of GA1.

Interaction of Glutaric Aciduria Type 1-Related glutaryl-CoA Dehydrogenase with Mitochondrial Matrix Proteins

PubMed Central

Schmiesing, Jessica; Schlüter, Hartmut; Ullrich, Kurt; Braulke, Thomas; Mühlhausen, Chris

2014-01-01

Glutaric aciduria type 1 (GA1) is an inherited neurometabolic disorder caused by mutations in the GCDH gene encoding glutaryl-CoA dehydrogenase (GCDH), which forms homo- and heteromeric complexes in the mitochondrial matrix. GA1 patients are prone to the development of encephalopathic crises which lead to an irreversible disabling dystonic movement disorder. The clinical and biochemical manifestations of GA1 vary considerably and lack correlations to the genotype. Using an affinity chromatography approach we report here for the first time on the identification of mitochondrial proteins interacting directly with GCDH. Among others, dihydrolipoamide S-succinyltransferase (DLST) involved in the formation of glutaryl-CoA, and the β-subunit of the electron transfer flavoprotein (ETFB) serving as electron acceptor, were identified as GCDH binding partners. We have adapted the yellow fluorescent protein-based fragment complementation assay and visualized the oligomerization of GCDH as well as its direct interaction with DLST and ETFB in mitochondria of living cells. These data suggest that GCDH is a constituent of multimeric mitochondrial dehydrogenase complexes, and the characterization of their interrelated functions may provide new insights into the regulation of lysine oxidation and the pathophysiology of GA1. PMID:24498361

Architecture of the ParF*ParG protein complex involved in prokaryotic DNA segregation.

PubMed

Barillà, Daniela; Hayes, Finbarr

2003-07-01

The mechanism by which low copy number plasmids are segregated at cell division involves the concerted action of two plasmid-encoded proteins that assemble on a centromere-like site. This study explores the topology of the DNA segregation machinery specified by the parFG locus of TP228, a partition system which is phylogenetically distinct from more well-characterized archetypes. A variety of genetic, biochemical and biophysical strategies revealed that the ParG protein is dimeric. ParF, which is more closely related to the cell division regulator MinD than to the prototypical ParA partition protein of plasmid P1, is instead multimeric and its polymeric state appears to be modulated by ATP which correlates with the proposed ATP-binding activity of ParF. ParG interacts in a sequence-specific manner with the DNA region upstream of the parFG locus and this binding is modulated by ParF. Intriguingly, the ParF and ParG proteins form at least two types of discrete complex in the absence of this region suggesting that the assembly dynamics of these proteins onto DNA is intricate.

The molecular genetics of von Willebrand disease.

PubMed

Berber, Ergül

2012-12-01

Quantitative and/or qualitative deficiency of von Willebrand factor (vWF) is associated with the most common inherited bleeding disease von Willebrand disease (vWD). vWD is a complex disease with clinical and genetic heterogeneity. Incomplete penetrance and variable expression due to genetic and environmental factors contribute to its complexity. vWD also has a complex molecular pathogenesis. Some vWF gene mutations are associated with the affected vWF biosynthesis and multimerization, whereas others are associated with increased clearance and functional impairment. Moreover, in addition to a particular mutation, type O blood may result in the more severe phenotype. The present review aimed to provide a summary of the current literature on the molecular genetics of vWD. None declared.

Alterations in Peptidoglycan Cross-Linking Suppress the Secretin Assembly Defect Caused by Mutation of GspA in the Type II Secretion System.

PubMed

Vanderlinde, Elizabeth M; Strozen, Timothy G; Hernández, Sara B; Cava, Felipe; Howard, S Peter

2017-04-15

In Gram-negative bacteria, the peptidoglycan (PG) cell wall is a significant structural barrier for outer membrane protein assembly. In Aeromonas hydrophila , outer membrane multimerization of the type II secretion system (T2SS) secretin ExeD requires the function of the inner membrane assembly factor complex ExeAB. The putative mechanism of the complex involves the reorganization of PG and localization of ExeD, whereby ExeA functions by interacting with PG to form a site for secretin assembly and ExeB forms an interaction with ExeD. This mechanism led us to hypothesize that increasing the pore size of PG would circumvent the requirement for ExeA in the assembly of the ExeD secretin. Growth of A. hydrophila in 270 mM Gly reduced PG cross-links by approximately 30% and led to the suppression of secretin assembly defects in exeA strains and in those expressing ExeA mutants by enabling localization of the secretin in the outer membrane. We also established a heterologous ExeD assembly system in Escherichia coli and showed that ExeAB and ExeC are the only A. hydrophila proteins required for the assembly of the ExeD secretin in E. coli and that ExeAB-independent assembly of ExeD can occur upon overexpression of the d,d-carboxypeptidase PBP 5. These results support an assembly model in which, upon binding to PG, ExeA induces multimerization and pore formation in the sacculus, which enables ExeD monomers to interact with ExeB and assemble into a secretin that both is inserted in the outer membrane and crosses the PG layer to interact with the inner membrane platform of the T2SS. IMPORTANCE The PG layer imposes a strict structural impediment for the assembly of macromolecular structures that span the cell envelope and serve as virulence factors in Gram-negative species. This work revealed that by decreasing PG cross-linking by growth in Gly, the absolute requirement for the PG-binding activity of ExeA in the assembly of the ExeD secretin was alleviated in A. hydrophila In a heterologous assembly model in E. coli , expression of the carboxypeptidase PBP 5 could relieve the requirement for ExeAB in the assembly of the ExeD secretin. These results provide some mechanistic details of the ExeAB assembly complex function, in which the PG-binding and oligomerization functions of ExeAB are used to create a pore in the PG that is required for secretin assembly. Copyright © 2017 American Society for Microbiology.

Investigation of the function of the putative self-association site of Epstein-Barr virus (EBV) glycoprotein 42 (gp42)

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

Rowe, Cynthia L., E-mail: c-rowe@northwestern.edu; Matsuura, Hisae, E-mail: hisaem@stanford.edu; Interdepartmental Biological Sciences Program, Northwestern University, Evanston, IL 60208

The Epstein-Barr virus (EBV) glycoprotein 42 (gp42) is a type II membrane protein essential for entry into B cells but inhibits entry into epithelial cells. X-ray crystallography suggests that gp42 may form dimers when bound to human leukocyte antigen (HLA) class II receptor (Mullen et al., 2002) or multimerize when not bound to HLA class II (Kirschner et al., 2009). We investigated this self-association of gp42 using several different approaches. We generated soluble mutants of gp42 containing mutations within the self-association site and found that these mutants have a defect in fusion. The gp42 mutants bound to gH/gL and HLAmore » class II, but were unable to bind wild-type gp42 or a cleavage mutant of gp42. Using purified gp42, gH/gL, and HLA, we found these proteins associate 1:1:1 by gel filtration suggesting that gp42 dimerization or multimerization does not occur or is a transient event undetectable by our methods.« less

Hydrogenase polypeptide and methods of use

DOEpatents

Adams, Michael W.W.; Hopkins, Robert C.; Jenney, JR, Francis E.; Sun, Junsong

2016-02-02

Provided herein are polypeptides having hydrogenase activity. The polypeptide may be multimeric, and may have hydrogenase activity of at least 0.05 micromoles H.sub.2 produced min.sup.-1 mg protein.sup.-1. Also provided herein are polynucleotides encoding the polypeptides, genetically modified microbes that include polynucleotides encoding one or more subunits of the multimeric polypeptide, and methods for making and using the polypeptides.

The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers

DOE PAGES

Olek, Anna T.; Rayon, Catherine; Makowski, Lee; ...

2014-07-10

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice ( Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain,more » elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. As a result, the arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.« less

A Novel RNA Polymerase I Transcription Initiation Factor, TIF-IE, Commits rRNA Genes by Interaction with TIF-IB, Not by DNA Binding

PubMed Central

Al-Khouri, Anna Maria; Paule, Marvin R.

2002-01-01

In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE. PMID:11784852

A novel RNA polymerase I transcription initiation factor, TIF-IE, commits rRNA genes by interaction with TIF-IB, not by DNA binding.

PubMed

Al-Khouri, Anna Maria; Paule, Marvin R

2002-02-01

In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE.

The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers.

PubMed

Olek, Anna T; Rayon, Catherine; Makowski, Lee; Kim, Hyung Rae; Ciesielski, Peter; Badger, John; Paul, Lake N; Ghosh, Subhangi; Kihara, Daisuke; Crowley, Michael; Himmel, Michael E; Bolin, Jeffrey T; Carpita, Nicholas C

2014-07-01

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. The arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize. © 2014 American Society of Plant Biologists. All rights reserved.

Aggregate complexes of HIV-1 induced by multimeric antibodies.

PubMed

Stieh, Daniel J; King, Deborah F; Klein, Katja; Liu, Pinghuang; Shen, Xiaoying; Hwang, Kwan Ki; Ferrari, Guido; Montefiori, David C; Haynes, Barton; Pitisuttithum, Punnee; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Rerks-Ngarm, Supachai; Michael, Nelson L; Robb, Merlin L; Kim, Jerome H; Denny, Thomas N; Tomaras, Georgia D; Shattock, Robin J

2014-10-02

Antibody mediated viral aggregation may impede viral transfer across mucosal surfaces by hindering viral movement in mucus, preventing transcytosis, or reducing inter-cellular penetration of epithelia thereby limiting access to susceptible mucosal CD4 T cells and dendritic cells. These functions may work together to provide effective immune exclusion of virus from mucosal tissue; however little is known about the antibody characteristics required to induce HIV aggregation. Such knowledge may be critical to the design of successful immunization strategies to facilitate viral immune exclusion at the mucosal portals of entry. The potential of neutralizing and non-neutralizing IgG and IgA monoclonals (mAbs) to induce HIV-1 aggregation was assessed by Dynamic light scattering (DLS). Although neutralizing and non-neutralizing IgG mAbs and polyclonal HIV-Ig efficiently aggregated soluble Env trimers, they were not capable of forming viral aggregates. In contrast, dimeric (but not monomeric) IgA mAbs induced stable viral aggregate populations that could be separated from uncomplexed virions. Epitope specificity influenced both the degree of aggregation and formation of higher order complexes by dIgA. IgA purified from serum of uninfected RV144 vaccine trial responders were able to efficiently opsonize viral particles in the absence of significant aggregation, reflective of monomeric IgA. These results collectively demonstrate that dIgA is capable of forming stable viral aggregates providing a plausible basis for testing the effectiveness of aggregation as a potential protection mechanism at the mucosal portals of viral entry.

Transformation of glucocorticoid receptors bound to the antagonist RU 486: Effects of alkaline phosphatase

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

Gruol, D.J.; Wolfe, K.A.

1990-08-28

RU 486 is a synthetic steroid that binds avidly to glucocorticoid receptors without promoting their transformation into activated transcription factors. A significant part of this behavior has been shown to be due to a failure of the RU 486 bound receptor to be efficiently released from a larger (sedimenting at 8-9 S) multimeric complex containing the 90-kDa heat shock protein. The studies have found that in vitro at 15{degree}C the RU 486-receptor was slowly released from the 8-9S complex and converted into a DNA binding protein by a process that could be blocked by sodium fluoride. Moreover, this transition wasmore » significantly accelerated by treatment with alkaline phosphatase. High-resolution anion-exchange chromatography showed that the profile of receptor subspecies released from the 8-9S complex was different for the RU 486 bound receptor when compared to the receptor occupied by the agonist triamcinolone acetonide. Production of the earliest eluting receptor form (peak A) was inhibited with RU 486. Treatment of the Ru 486-receptor with alkaline phosphatase increased the formation of the peak A subspecies as well as the capacity of receptor to bind DNA-cellulose. Taken together, the results indicate that phosphorylation of the receptor or a tightly bound factor contributes to defining the capacity with which individual steroids can promote dissociation of the 8-9S complex and conversion of the glucocorticoid receptor into a DNA-binding protein.« less

Occurrence of a multimeric high-molecular-weight glyceraldehyde-3-phosphate dehydrogenase in human serum.

PubMed

Kunjithapatham, Rani; Geschwind, Jean-Francois; Devine, Lauren; Boronina, Tatiana N; O'Meally, Robert N; Cole, Robert N; Torbenson, Michael S; Ganapathy-Kanniappan, Shanmugasundaram

2015-04-03

Cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phylogenetically conserved, ubiquitous enzyme that plays an indispensable role in energy metabolism. Although a wealth of information is available on cellular GAPDH, there is a clear paucity of data on its extracellular counterpart (i.e., the secreted or extracellular GAPDH). Here, we show that the extracellular GAPDH in human serum is a multimeric, high-molecular-weight, yet glycolytically active enzyme. The high-molecular-weight multimers of serum GAPDH were identified by immunodetection on one- and two-dimensional gel electrophoresis using multiple antibodies specific for various epitopes of GAPDH. Partial purification of serum GAPDH by DEAE Affigel affinity/ion exchange chromatography further established the multimeric composition of serum GAPDH. In vitro data demonstrated that human cell lines secrete a multimeric, high-molecular-weight enzyme similar to that of serum GAPDH. Furthermore, LC-MS/MS analysis of extracellular GAPDH from human cell lines confirmed the presence of unique peptides of GAPDH in the high-molecular-weight subunits. Furthermore, data from pulse-chase experiments established the presence of high-molecular-weight subunits in the secreted, extracellular GAPDH. Taken together, our findings demonstrate the presence of a high-molecular-weight, enzymatically active secretory GAPDH in human serum that may have a hitherto unknown function in humans.

Homotypic Interaction of Bunyamwera Virus Nucleocapsid Protein

PubMed Central

Leonard, Vincent H. J.; Kohl, Alain; Osborne, Jane C.; McLees, Angela; Elliott, Richard M.

2005-01-01

The bunyavirus nucleocapsid protein, N, plays a central role in viral replication in encapsidating the three genomic RNA segments to form functional templates for transcription and replication by the viral RNA-dependent RNA polymerase. Here we report functional mapping of interacting domains of the Bunyamwera orthobunyavirus N protein by yeast and mammalian two-hybrid systems, immunoprecipitation experiments, and chemical cross-linking studies. N forms a range of multimers from dimers to high-molecular-weight structures, independently of the presence of RNA. Deletion of the N- or C-terminal domains resulted in loss of activity in a minireplicon assay and a decreased capacity for N to form higher multimers. Our data suggest a head-to-head and tail-to-tail multimerization model for the orthobunyavirus N protein. PMID:16189017

Mechanical Activation of a Multimeric Adhesive Protein Through Domain Conformational Change

NASA Astrophysics Data System (ADS)

Wijeratne, Sithara S.; Botello, Eric; Yeh, Hui-Chun; Zhou, Zhou; Bergeron, Angela L.; Frey, Eric W.; Patel, Jay M.; Nolasco, Leticia; Turner, Nancy A.; Moake, Joel L.; Dong, Jing-fei; Kiang, Ching-Hwa

2013-03-01

The mechanical force-induced activation of the adhesive protein von Willebrand factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (PVWF) multimers to bind platelets. Here, we showed that a pathological level of high shear stress exposure of PVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of the multimeric VWF. We found that shear-activated PVWF multimers are more resistant to mechanical unfolding than nonsheared PVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of PVWF multimers.

Molecular switch-like regulation in motor proteins.

PubMed

Tafoya, Sara; Bustamante, Carlos

2018-06-19

Motor proteins are powered by nucleotide hydrolysis and exert mechanical work to carry out many fundamental biological tasks. To ensure their correct and efficient performance, the motors' activities are allosterically regulated by additional factors that enhance or suppress their NTPase activity. Here, we review two highly conserved mechanisms of ATP hydrolysis activation and repression operating in motor proteins-the glutamate switch and the arginine finger-and their associated regulatory factors. We examine the implications of these regulatory mechanisms in proteins that are formed by multiple ATPase subunits. We argue that the regulatory mechanisms employed by motor proteins display features similar to those described in small GTPases, which require external regulatory elements, such as dissociation inhibitors, exchange factors and activating proteins, to switch the protein's function 'on' and 'off'. Likewise, similar regulatory roles are taken on by the motor's substrate, additional binding factors, and even adjacent subunits in multimeric complexes. However, in motor proteins, more than one regulatory factor and the two mechanisms described here often underlie the machine's operation. Furthermore, ATPase regulation takes place throughout the motor's cycle, which enables a more complex function than the binary 'active' and 'inactive' states.This article is part of a discussion meeting issue 'Allostery and molecular machines'. © 2018 The Author(s).

PDZ Ligand Binding-Induced Conformational Coupling of the PDZ-SH3-GK Tandems in PSD-95 Family MAGUKs.

PubMed

Zeng, Menglong; Ye, Fei; Xu, Jia; Zhang, Mingjie

2018-01-05

Discs large (DLG) MAGUKs are abundantly expressed in glutamatergic synapses, crucial for synaptic transmission, and plasticity by anchoring various postsynaptic components including glutamate receptors, downstream scaffold proteins and signaling enzymes. Different DLG members have shared structures and functions, but also contain unique features. How DLG family proteins function individually and cooperatively is largely unknown. Here, we report that PSD-95 PDZ3 directly couples with SH3-GK tandem in a PDZ ligand binding-dependent manner, and the coupling can promote PSD-95 dimerization and multimerization. Aided by sortase-mediated protein ligation and selectively labeling, we elucidated the PDZ3/SH3-GK conformational coupling mechanism using NMR spectroscopy. We further demonstrated that PSD-93, but not SAP102, can also undergo PDZ3 ligand binding-induced conformational coupling with SH3-GK and form homo-oligomers. Interestingly, PSD-95 and PSD-93 can also form ligand binding-induced hetero-oligomers, suggesting a cooperative assembly mechanism for the mega-N-methyl-d-aspartate receptor synaptic signaling complex. Finally, we provide evidence showing that ligand binding-induced conformational coupling between PDZ and SH3-GK is a common feature for other MAGUKs including CASK and PALS1. Copyright © 2017 Elsevier Ltd. All rights reserved.

A photo-cross-linking approach to monitor folding and assembly of newly synthesized proteins in a living cell.

PubMed

Miyazaki, Ryoji; Myougo, Naomi; Mori, Hiroyuki; Akiyama, Yoshinori

2018-01-12

Many proteins form multimeric complexes that play crucial roles in various cellular processes. Studying how proteins are correctly folded and assembled into such complexes in a living cell is important for understanding the physiological roles and the qualitative and quantitative regulation of the complex. However, few methods are suitable for analyzing these rapidly occurring processes. Site-directed in vivo photo-cross-linking is an elegant technique that enables analysis of protein-protein interactions in living cells with high spatial resolution. However, the conventional site-directed in vivo photo-cross-linking method is unsuitable for analyzing dynamic processes. Here, by combining an improved site-directed in vivo photo-cross-linking technique with a pulse-chase approach, we developed a new method that can analyze the folding and assembly of a newly synthesized protein with high spatiotemporal resolution. We demonstrate that this method, named the pulse-chase and in vivo photo-cross-linking experiment (PiXie), enables the kinetic analysis of the formation of an Escherichia coli periplasmic (soluble) protein complex (PhoA). We also used our new technique to investigate assembly/folding processes of two membrane complexes (SecD-SecF in the inner membrane and LptD-LptE in the outer membrane), which provided new insights into the biogenesis of these complexes. Our PiXie method permits analysis of the dynamic behavior of various proteins and enables examination of protein-protein interactions at the level of individual amino acid residues. We anticipate that our new technique will have valuable utility for studies of protein dynamics in many organisms. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Subunit stoichiometry of human muscle chloride channels.

PubMed

Fahlke, C; Knittle, T; Gurnett, C A; Campbell, K P; George, A L

1997-01-01

Voltage-gated Cl- channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl- channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their native configurations exhibit similar sedimentation properties consistent with a multimeric complex having a molecular mass of a dimer. Expression of the heterodimeric channel in a mammalian cell line results in a homogenous population of Cl- channels exhibiting novel gating properties that are best explained by the formation of heteromultimeric channels with an even number of subunits. Heteromultimeric channels were not evident in cells cotransfected with homodimeric WT-WT and D136G-D136G constructs excluding the possibility that functional hClC-1 channels are assembled from more than two subunits. These results demonstrate that the functional hClC-1 unit consists of two subunits.

Crystal Structure of the Nipah Virus Phosphoprotein Tetramerization Domain

PubMed Central

Bruhn, Jessica F.; Barnett, Katherine C.; Bibby, Jaclyn; Thomas, Jens M. H.; Keegan, Ronan M.; Rigden, Daniel J.; Bornholdt, Zachary A.

2014-01-01

The Nipah virus phosphoprotein (P) is multimeric and tethers the viral polymerase to the nucleocapsid. We present the crystal structure of the multimerization domain of Nipah virus P: a long, parallel, tetrameric, coiled coil with a small, α-helical cap structure. Across the paramyxoviruses, these domains share little sequence identity yet are similar in length and structural organization, suggesting a common requirement for scaffolding or spatial organization of the functions of P in the virus life cycle. PMID:24155387

Integrated fluorescence correlation spectroscopy device for point-of-care clinical applications

PubMed Central

Olson, Eben; Torres, Richard; Levene, Michael J.

2013-01-01

We describe an optical system which reduces the cost and complexity of fluorescence correlation spectroscopy (FCS), intended to increase the suitability of the technique for clinical use. Integration of the focusing optics and sample chamber into a plastic component produces a design which is simple to align and operate. We validate the system by measurements on fluorescent dye, and compare the results to a commercial instrument. In addition, we demonstrate its application to measurements of concentration and multimerization of the clinically relevant protein von Willebrand factor (vWF) in human plasma. PMID:23847733

Rational assembly of nanoparticle superlattices with designed lattice symmetries

DOEpatents

Gang, Oleg; Lu, Fang; Tagawa, Miho

2017-09-05

A method for lattice design via multivalent linkers (LDML) is disclosed that introduces a rationally designed symmetry of connections between particles in order to achieve control over the morphology of their assembly. The method affords the inclusion of different programmable interactions within one linker that allow an assembly of different types of particles. The designed symmetry of connections is preferably provided utilizing DNA encoding. The linkers may include fabricated "patchy" particles, DNA scaffold constructs and Y-shaped DNA linkers, anisotropic particles, which are preferably functionalized with DNA, multimeric protein-DNA complexes, and particles with finite numbers of DNA linkers.

DYNLL/LC8 Protein Controls Signal Transduction through the Nek9/Nek6 Signaling Module by Regulating Nek6 Binding to Nek9

PubMed Central

Regué, Laura; Sdelci, Sara; Bertran, M. Teresa; Caelles, Carme; Reverter, David; Roig, Joan

2011-01-01

The NIMA family protein kinases Nek9/Nercc1 and the highly similar Nek6 and Nek7 form a signaling module activated in mitosis, when they are involved in the control of spindle organization and function. Here we report that Nek9, the module upstream kinase, binds to DYNLL/LC8, a highly conserved protein originally described as a component of the dynein complex. LC8 is a dimer that interacts with different proteins and has been suggested to act as a dimerization hub promoting the organization and oligomerization of partially disorganized partners. We find that the interaction of LC8 with Nek9 depends on a (K/R)XTQT motif adjacent to the Nek9 C-terminal coiled coil motif, results in Nek9 multimerization, and increases the rate of Nek9 autoactivation. LC8 binding to Nek9 is regulated by Nek9 activity through the autophosphorylation of Ser944, a residue immediately N-terminal to the (K/R)XTQT motif. Remarkably, LC8 binding interferes with the interaction of Nek9 with its downstream partner Nek6 as well as with Nek6 activation, thus controlling both processes. Our work sheds light into the control of signal transduction through the module formed by Nek9 and Nek6/7 and uncovers a novel manner in which LC8 can regulate partner physiology by interfering with protein complex formation. We suggest that this and other LC8 functions can be specifically regulated by partner phosphorylation. PMID:21454704

Discovery of the type VII ESX-1 secretion needle?

PubMed

Ates, Louis S; Brosch, Roland

2017-01-01

Mycobacterium tuberculosis, the etiological agent of human tuberculosis, harbours five ESAT-6/type VII secretion (ESX/T7S) systems. The first esx gene clusters were identified during the genome-sequencing project of M. tuberculosis H37Rv. Follow-up studies revealed additional genes playing important roles in ESX/T7S systems. Among the latter genes, one can find those that encode Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins as well as a gene cluster that is encoded >260 kb upstream of the esx-1 locus and encodes ESX-1 secretion-associated proteins EspA (Rv3616c), EspC (Rv3615c) and EspD (Rv3614c). The espACD cluster has been suggested to have an important function in ESX-1 secretion since EspA-EspC and EsxA-EsxB are mutually co-dependent on each other for secretion. However, the molecular mechanism of this co-dependence and interaction between the substrates remained unknown. In this issue of Molecular Microbiology, Lou and colleagues show that EspC forms high-molecular weight polymerization complexes that resemble selected components of type II, III and/or IV secretion systems of Gram-negative bacteria. Indeed, EspC-multimeric complexes form filamentous structures that could well represent a secretion needle of ESX-1 type VII secretion systems. This exciting observation opens new avenues for research to discover and characterize ESX/T7S components and elucidates the co-dependence of EsxA/B secretion with EspA/C. © 2016 John Wiley & Sons Ltd.

Molecular recognition of the Tes LIM2-3 domains by the actin-related protein Arp7A.

PubMed

Boëda, Batiste; Knowles, Phillip P; Briggs, David C; Murray-Rust, Judith; Soriano, Erika; Garvalov, Boyan K; McDonald, Neil Q; Way, Michael

2011-04-01

Actin-related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized "orphan" Arps, which appear to be mostly testis-specific. Here we show that Arp7A, which has 43% sequence identity with β-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65-residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1-65(Arp7A)·LIM2-3(Tes)·EVH1(Mena) complex reveals that residues 28-49 of Arp7A contact the LIM2-3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2-3 junction are critical for the Arp7A-Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.

Evaluation of protein docking predictions using Hex 3.1 in CAPRI rounds 1 and 2.

PubMed

Ritchie, David W

2003-07-01

This article describes and reviews our efforts using Hex 3.1 to predict the docking modes of the seven target protein-protein complexes presented in the CAPRI (Critical Assessment of Predicted Interactions) blind docking trial. For each target, the structure of at least one of the docking partners was given in its unbound form, and several of the targets involved large multimeric structures (e.g., Lactobacillus HPr kinase, hemagglutinin, bovine rotavirus VP6). Here we describe several enhancements to our original spherical polar Fourier docking correlation algorithm. For example, a novel surface sphere smothering algorithm is introduced to generate multiple local coordinate systems around the surface of a large receptor molecule, which may be used to define a small number of initial ligand-docking orientations distributed over the receptor surface. High-resolution spherical polar docking correlations are performed over the resulting receptor surface patches, and candidate docking solutions are refined by using a novel soft molecular mechanics energy minimization procedure. Overall, this approach identified two good solutions at rank 5 or less for two of the seven CAPRI complexes. Subsequent analysis of our results shows that Hex 3.1 is able to place good solutions within a list of

Plasma proteins of rainbow trout (Oncorhynchus mykiss) isolated by binding to lipopolysaccharide from Aeromonas salmonicida.

PubMed

Hoover, G J; el-Mowafi, A; Simko, E; Kocal, T E; Ferguson, H W; Hayes, M A

1998-07-01

In an attempt to find plasma proteins that might be involved in the constitutive resistance of rainbow trout to furunculosis, a disease caused by Aeromonas salmonicida (AS), we purified serum and plasma proteins based on their calcium- and carbohydrate-dependent affinity for A. salmonicida lipopolysaccharide (LPS) coupled to an epoxy-activated synthetic matrix (Toyopearl AF Epoxy 650M). A multimeric family of high molecular weight (96 to 200-kDa) LPS-binding proteins exhibiting both calcium and mannose dependent binding was isolated. Upon reduction the multimers collapsed to subunits of approximately 16-kDa as estimated by 1D-PAGE and exhibited pI values of 5.30 and 5.75 as estimated from 2D-PAGE. Their N-terminal sequences were related to rainbow trout ladderlectin (RT-LL), a Sepharose-binding protein. Polyclonal antibodies to the LPS-purified 16-kDa subunits recognized both the reduced 16-kDa subunits and the non-reduced multimeric forms. A calcium- and N-acetylglucosamine (GlcNAc)-dependent LPS-binding multimeric protein (approximately 207-kDa) composed of 34.5-kDa subunits was purified and found to be identical to trout serum amyloid P (SAP) by N-terminal sequence (DLQDLSGKVFV). A protein of 24-kDa, in reduced and non-reduced conditions, was isolated and had N-terminal sequence identity with a known C-reactive protein (CRP) homologue, C-polysaccharide-binding protein 2 (TCBP2) of rainbow trout. A novel calcium-dependent LPS-binding protein was purified and termed rainbow trout lectin 37 (RT-L37). This protein, composed of dimers, tetramers and pentamers of 37 kDa subunits (pI 5.50-6.10) with N-terminal sequence (IQE(D/N)GHAEAPGATTVLNEILR) showed no close homology to proteins known or predicted from cDNA sequences. These findings demonstrate that rainbow trout have several blood proteins with lectin properties for the LPS of A. salmonicida; the biological functions of these proteins in resistance to furunculosis are still unknown.

Surfactant Protein D in Respiratory and Non-Respiratory Diseases

PubMed Central

Sorensen, Grith L.

2018-01-01

Surfactant protein D (SP-D) is a multimeric collectin that is involved in innate immune defense and expressed in pulmonary, as well as non-pulmonary, epithelia. SP-D exerts antimicrobial effects and dampens inflammation through direct microbial interactions and modulation of host cell responses via a series of cellular receptors. However, low protein concentrations, genetic variation, biochemical modification, and proteolytic breakdown can induce decomposition of multimeric SP-D into low-molecular weight forms, which may induce pro-inflammatory SP-D signaling. Multimeric SP-D can decompose into trimeric SP-D, and this process, and total SP-D levels, are partly determined by variation within the SP-D gene, SFTPD. SP-D has been implicated in the development of respiratory diseases including respiratory distress syndrome, bronchopulmonary dysplasia, allergic asthma, and chronic obstructive pulmonary disease. Disease-induced breakdown or modifications of SP-D facilitate its systemic leakage from the lung, and circulatory SP-D is a promising biomarker for lung injury. Moreover, studies in preclinical animal models have demonstrated that local pulmonary treatment with recombinant SP-D is beneficial in these diseases. In recent years, SP-D has been shown to exert antimicrobial and anti-inflammatory effects in various non-pulmonary organs and to have effects on lipid metabolism and pro-inflammatory effects in vessel walls, which enhance the risk of atherosclerosis. A common SFTPD polymorphism is associated with atherosclerosis and diabetes, and SP-D has been associated with metabolic disorders because of its effects in the endothelium and adipocytes and its obesity-dampening properties. This review summarizes and discusses the reported genetic associations of SP-D with disease and the clinical utility of circulating SP-D for respiratory disease prognosis. Moreover, basic research on the mechanistic links between SP-D and respiratory, cardiovascular, and metabolic diseases is summarized. Perspectives on the development of SP-D therapy are addressed. PMID:29473039

Synthesis and evaluation of novel multimeric neurotensin(8-13) analogs.

PubMed

Hultsch, Christina; Pawelke, Beate; Bergmann, Ralf; Wuest, Frank

2006-09-01

Neurotensin(8-13) is a hexapeptide with subnanomolar affinity to the neurotensin receptor 1 which is expressed with high incidence in several human tumor entities. Thus, radiolabeled neurotensin(8-13) might be used for tumor targeting. However, its application is limited by insufficient metabolic stability. The present study aims at improving metabolic stability by the synthesis of multimeric neurotensin(8-13) derivatives rather than commonly employed chemical modifications of the peptide itself. Thus, different dimeric and tetrameric peptides carrying C- or N-terminal attached neurotensin(8-13) moieties have been synthesized and their binding affinity toward the neurotensin receptor has been determined. The results demonstrate that branched compounds containing neurotensin(8-13) attached via its C-terminus only show low receptor affinities, whilst derivatives with neurotensin(8-13) attached via the N-terminus show IC50 values in the nanomolar range. Moreover, within the multimeric neurotensin(8-13) derivatives with neurotensin(8-13) attached via the N-terminus an increasing number of branching units lead to higher binding affinities toward the neurotensin receptor.

Assessment of Protein Side-Chain Conformation Prediction Methods in Different Residue Environments

PubMed Central

Peterson, Lenna X.; Kang, Xuejiao; Kihara, Daisuke

2016-01-01

Computational prediction of side-chain conformation is an important component of protein structure prediction. Accurate side-chain prediction is crucial for practical applications of protein structure models that need atomic detailed resolution such as protein and ligand design. We evaluated the accuracy of eight side-chain prediction methods in reproducing the side-chain conformations of experimentally solved structures deposited to the Protein Data Bank. Prediction accuracy was evaluated for a total of four different structural environments (buried, surface, interface, and membrane-spanning) in three different protein types (monomeric, multimeric, and membrane). Overall, the highest accuracy was observed for buried residues in monomeric and multimeric proteins. Notably, side-chains at protein interfaces and membrane-spanning regions were better predicted than surface residues even though the methods did not all use multimeric and membrane proteins for training. Thus, we conclude that the current methods are as practically useful for modeling protein docking interfaces and membrane-spanning regions as for modeling monomers. PMID:24619909

Tandem-multimeric F3-gelonin fusion toxins for enhanced anti-cancer activity for prostate cancer treatment.

PubMed

Shin, Meong Cheol; Min, Kyoung Ah; Cheong, Heesun; Moon, Cheol; Huang, Yongzhuo; He, Huining; Yang, Victor C

2017-05-30

Despite significant progress in prostate cancer treatment, yet, it remains the leading diagnosed cancer and is responsible for high incidence of cancer related deaths in the U.S. Because of the insufficient efficacy of small molecule anti-cancer drugs, significant interest has been drawn to more potent macromolecular agents such as gelonin, a plant-derived ribosome inactivating protein (RIP) that efficiently inhibits protein translation. However, in spite of the great potency to kill tumor cells, gelonin lacks ability to internalize tumor cells and furthermore, cannot distinguish between tumor and normal cells. To address this challenge, we genetically engineered gelonin fusion proteins with varied numbers of F3 peptide possessing homing ability to various cancer cells and angiogenic blood vessels. The E. coli produced F3-gelonin fusion proteins possessed equipotent activity to inhibit protein translation in cell-free protein translation systems to unmodified gelonin; however, they displayed higher cell uptake that led to significantly augmented cytotoxicity. Compared with gelonin fusion with one F3 peptide (F3-Gel), tandem-multimeric F3-gelonins showed even greater cell internalization and tumor cell killing ability. Moreover, when tested against LNCaP s.c. xenograft tumor bearing mice, more significant tumor growth inhibition was observed from the mice treated with tandem-multimeric F3-gelonins. Overall, this research demonstrated the potential of utilizing tandem multimeric F3-modified gelonin as highly effective anticancer agents to overcome the limitations of current chemotherapeutic drugs. Copyright © 2017. Published by Elsevier B.V.

Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore.

PubMed

Panjwani, Anusha; Strauss, Mike; Gold, Sarah; Wenham, Hannah; Jackson, Terry; Chou, James J; Rowlands, David J; Stonehouse, Nicola J; Hogle, James M; Tuthill, Tobias J

2014-08-01

Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

Methylation-regulated decommissioning of multimeric PP2A complexes

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

Wu, Cheng-Guo; Zheng, Aiping; Jiang, Li

2017-12-01

Dynamic assembly/disassembly of signaling complexes are crucial for cellular functions. Specialized latency and activation chaperones control the biogenesis of protein phosphatase 2A (PP2A) holoenzymes that contain a common scaffold and catalytic subunits and a variable regulatory subunit. Here we show that the butterfly-shaped TIPRL (TOR signaling pathway regulator) makes highly integrative multibranching contacts with the PP2A catalytic subunit, selective for the unmethylated tail and perturbing/inactivating the phosphatase active site. TIPRL also makes unusual wobble contacts with the scaffold subunit, allowing TIPRL, but not the overlapping regulatory subunits, to tolerate disease-associated PP2A mutations, resulting in reduced holoenzyme assembly and enhanced inactivationmore » of mutant PP2A. Strikingly, TIPRL and the latency chaperone, α4, coordinate to disassemble active holoenzymes into latent PP2A, strictly controlled by methylation. Our study reveals a mechanism for methylation-responsive inactivation and holoenzyme disassembly, illustrating the complexity of regulation/signaling, dynamic complex disassembly, and disease mutations in cancer and intellectual disability.« less

Control of developmentally primed erythroid genes by combinatorial co-repressor actions

PubMed Central

Stadhouders, Ralph; Cico, Alba; Stephen, Tharshana; Thongjuea, Supat; Kolovos, Petros; Baymaz, H. Irem; Yu, Xiao; Demmers, Jeroen; Bezstarosti, Karel; Maas, Alex; Barroca, Vilma; Kockx, Christel; Ozgur, Zeliha; van Ijcken, Wilfred; Arcangeli, Marie-Laure; Andrieu-Soler, Charlotte; Lenhard, Boris; Grosveld, Frank; Soler, Eric

2015-01-01

How transcription factors (TFs) cooperate within large protein complexes to allow rapid modulation of gene expression during development is still largely unknown. Here we show that the key haematopoietic LIM-domain-binding protein-1 (LDB1) TF complex contains several activator and repressor components that together maintain an erythroid-specific gene expression programme primed for rapid activation until differentiation is induced. A combination of proteomics, functional genomics and in vivo studies presented here identifies known and novel co-repressors, most notably the ETO2 and IRF2BP2 proteins, involved in maintaining this primed state. The ETO2–IRF2BP2 axis, interacting with the NCOR1/SMRT co-repressor complex, suppresses the expression of the vast majority of archetypical erythroid genes and pathways until its decommissioning at the onset of terminal erythroid differentiation. Our experiments demonstrate that multimeric regulatory complexes feature a dynamic interplay between activating and repressing components that determines lineage-specific gene expression and cellular differentiation. PMID:26593974

Detection of Mutant Huntingtin Aggregation Conformers and Modulation of SDS-Soluble Fibrillar Oligomers by Small Molecules

PubMed Central

Sontag, Emily Mitchell; Lotz, Gregor P.; Yang, Guocheng; Sontag, Christopher J.; Cummings, Brian J.; Glabe, Charles G.; Muchowski, Paul J.; Thompson, Leslie Michels

2012-01-01

The Huntington’s disease (HD) mutation leads to a complex process of Huntingtin (Htt) aggregation into multimeric species that eventually form visible inclusions in cytoplasm, nuclei and neuronal processes. One hypothesis is that smaller, soluble forms of amyloid proteins confer toxic effects and contribute to early cell dysfunction. However, analysis of mutant Htt aggregation intermediates to identify conformers that may represent toxic forms of the protein and represent potential drug targets remains difficult. We performed a detailed analysis of aggregation conformers in multiple in vitro, cell and ex vivo models of HD. Conformation-specific antibodies were used to identify and characterize aggregation species, allowing assessment of multiple conformers present during the aggregation process. Using a series of assays together with these antibodies, several forms could be identified. Fibrillar oligomers, defined as having a β-sheet rich conformation, are observed in vitro using recombinant protein and in protein extracts from cells in culture or mouse brain and shown to be globular, soluble and non-sedimentable structures. Compounds previously described to modulate visible inclusion body formation and reduce toxicity in HD models were also tested and consistently found to alter the formation of fibrillar oligomers. Interestingly, these compounds did not alter the rate of visible inclusion formation, indicating that fibrillar oligomers are not necessarily the rate limiting step of inclusion body formation. Taken together, we provide insights into the structure and formation of mutant Htt fibrillar oligomers that are modulated by small molecules with protective potential in HD models. PMID:24086178

Mechanism of the formation of the RecA-ssDNA nucleoprotein filament structure: a coarse-grained approach.

PubMed

Mukherjee, Goutam; Pal, Arumay; Levy, Yaakov

2017-11-21

In prokaryotes, the RecA protein catalyzes the repair and strand exchange of double-stranded DNA. RecA binds to single-stranded DNA (ssDNA) and forms a presynaptic complex in which the protein polymerizes around the ssDNA to form a right-handed helical nucleoprotein filament structure. In the present work, the mechanism for the formation of the RecA-ssDNA filament structure is modeled using coarse-grained molecular dynamics simulations. Information from the X-ray structure was used to model the protein itself but not its interactions; the interactions between the protein and the ssDNA were modeled solely by electrostatic, aromatic, and repulsive energies. For the present study, the monomeric, dimeric, and trimeric units of RecA and 4, 8, and 11 NT-long ssDNA, respectively, were studied. Our results indicate that monomeric RecA is not sufficient for nucleoprotein filament formation; rather, dimeric RecA is the elementary binding unit, with higher multimeric units of RecA facilitating filament formation. Our results reveal that loop region flexibility at the primary binding site of RecA is essential for it to bind the incoming ssDNA, that the aromatic residues present in the loop region play an important role in ssDNA binding, and that ATP may play a role in guiding the ssDNA by changing the electrostatic potential of the RecA protein.

Alterations in the Balance of Amyloid-β Protein Precursor Species in the Cerebrospinal Fluid of Alzheimer's Disease Patients.

PubMed

Lopez-Font, Inmaculada; Boix, Claudia P; Zetterberg, Henrik; Blennow, Kaj; Sáez-Valero, Javier

2017-01-01

We recently demonstrated that soluble forms of the amyloid-β protein precursor (sAβPP) assemble into multimeric complexes in cerebrospinal fluid (CSF), which contributes to the underestimation of specific sAβPP species when assessed by ELISA. To circumvent this issue, we analyzed by SDS-PAGE large fragments of sAβPP and their variants in the CSF from Alzheimer's disease (AD; n = 20) and control (n = 20) subjects, probing with specific antibodies against particular domains. Similar levels of sAβPPα and sAβPPβ protein were found in CSF samples from AD and controls, yet there appeared to be a shift in the balance of the soluble full-length AβPP (sAβPPf) species in AD samples, with a decrease in the proportion of the lower (∼100 kDa) band relative to the upper (∼120 kDa) band. Similar differences were observed in the contribution of the major KPI-immunoreactive AβPP species. CSF samples also displayed differences in the correlations of AβPP species with classical AD biomarkers, particularly with respect to the Aβ42 peptide. The differences reveal alterations that probably reflect pathophysiological changes in the brain.

N-acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia.

PubMed

Welch, M; Todd, D E; Whitehead, N A; McGowan, S J; Bycroft, B W; Salmond, G P

2000-02-15

Quorum sensing via an N-acyl homoserine lactone (HSL) pheromone controls the biosynthesis of a carbapenem antibiotic in Erwinia carotovora. Transcription of the carbapenem biosynthetic genes is dependent on the LuxR-type activator protein, CarR. Equilibrium binding of a range of HSL molecules, which are thought to activate CarR to bind to its DNA target sequence, was examined using fluorescence quenching, DNA bandshift analysis, limited proteolysis and reporter gene assays. CarR bound the most physiologically relevant ligand, N-(3-oxohexanoyl)-L-homoserine lactone, with a stoichiometry of two molecules of ligand per dimer of protein and a dissociation constant of 1.8 microM, in good agreement with the concentration of HSL required to activate carbapenem production in vivo. In the presence of HSL, CarR formed a very high molecular weight complex with its target DNA, indicating that the ligand causes the protein to multimerize. Chemical cross-linking analysis supported this interpretation. Our data show that the ability of a given HSL to facilitate CarR binding to its target DNA sequence is directly proportional to the affinity of the HSL for the protein.

A member of a new plant gene family encoding a meprin and TRAF homology (MATH) domain-containing protein is involved in restriction of long distance movement of plant viruses

PubMed Central

Cosson, Patrick; Sofer, Luc; Schurdi-Levraud, Valérie

2010-01-01

Restriction of long distance movement of several potyviruses in Arabidopsis thaliana is controlled by at least three dominant restricted TEV movement (RTM) genes, named RTM1, RTM2 and RTM3 and acts as a non-conventional resistance. RTM1 encodes a protein belonging to the jacalin family and RTM2 encodes a protein which has similarities to small heat shock proteins. The recent cloning of RTM3 which encodes a protein belonging to an unknown protein family of 29 members that has a meprin and TRAF homology (MATH) domain in its N-terminal region and a coiled-coil (CC) domain at its C-terminal end is an important breakthrough for a better understanding of this resistance process. Not only the third gene involved in this resistance has been identified and has allowed revealing a new gene family in plant but the discovery that the RTM3 protein interacts directly with RTM1 strongly suggests that the RTM proteins form a multimeric complex. However, these data also highlight striking similarities of the RTM resistance with the well known R-gene mediated resistance. PMID:20930558

Clostridial Binary Toxins: Basic Understandings that Include Cell Surface Binding and an Internal "Coup de Grâce".

PubMed

Stiles, Bradley G

2017-01-01

Clostridium species can make a remarkable number of different protein toxins, causing many diverse diseases in humans and animals. The binary toxins of Clostridium botulinum, C. difficile, C. perfringens, and C. spiroforme are one group of enteric-acting toxins that attack the actin cytoskeleton of various cell types. These enterotoxins consist of A (enzymatic) and B (cell binding/membrane translocation) components that assemble on the targeted cell surface or in solution, forming a multimeric complex. Once translocated into the cytosol via endosomal trafficking and acidification, the A component dismantles the filamentous actin-based cytoskeleton via mono-ADP-ribosylation of globular actin. Knowledge of cell surface receptors and how these usurped, host-derived molecules facilitate intoxication can lead to novel ways of defending against these clostridial binary toxins. A molecular-based understanding of the various steps involved in toxin internalization can also unveil therapeutic intervention points that stop the intoxication process. Furthermore, using these bacterial proteins as medicinal shuttle systems into cells provides intriguing possibilities in the future. The pertinent past and state-of-the-art present, regarding clostridial binary toxins, will be evident in this chapter.

Inflammasome activation and assembly at a glance.

PubMed

Malik, Ankit; Kanneganti, Thirumala-Devi

2017-12-01

Inflammasomes are multimeric protein complexes that typically comprise a sensor, an adaptor and the zymogen procaspase-1. An inflammasome assembles in response to a diverse range of pathogen-associated or danger-associated molecular patterns (PAMPs or DAMPs). The inflammasome platform leads to activation of caspase-1 through proximity-induced self-cleavage, which further induces maturation of interleukins 1β and 18 (IL-1β and IL-18) through proteolytic cleavage of pro-IL-1β and pro-IL-18. Activated caspase-1 also cleaves gasdermin D, which leads to a particular form of cell death called pyroptosis. Mutations in genes that encode inflammasome components are associated with many inflammatory disorders, and studies in the past decade have highlighted the importance of appropriate activation of the inflammasome in homeostasis and disease pathogenesis. Therefore, much attention is being paid to uncover the modulators and regulators of inflammasome assembly and pyroptosis. This Cell Science at a Glance article and accompanying poster outlines the concepts in the activation of inflammasome sensors and assembly of the inflammasome platform. We also discuss recent insights into the mechanisms of regulation of inflammasome activity and the induction of cell death by pyroptosis. © 2017. Published by The Company of Biologists Ltd.

Immunization With Fc-Based Recombinant Epstein–Barr Virus gp350 Elicits Potent Neutralizing Humoral Immune Response in a BALB/c Mice Model

PubMed Central

Zhao, Bingchun; Zhang, Xiao; Krummenacher, Claude; Song, Shuo; Gao, Ling; Zhang, Haojiong; Xu, Miao; Feng, Lin; Feng, Qisheng; Zeng, Musheng; Xu, Yuting; Zeng, Yixin

2018-01-01

Epstein–Barr virus (EBV) was the first human virus proved to be closely associated with tumor development, such as lymphoma, nasopharyngeal carcinoma, and EBV-associated gastric carcinoma. Despite many efforts to develop prophylactic vaccines against EBV infection and diseases, no candidates have succeeded in effectively blocking EBV infection in clinical trials. Previous investigations showed that EBV gp350 plays a pivotal role in the infection of B-lymphocytes. Nevertheless, using monomeric gp350 proteins as antigens has not been effective in preventing infection. Multimeric forms of the antigen are more potently immunogenic than monomers; however, the multimerization elements used in previous constructs are not approved for human clinical trials. To prepare a much-needed EBV prophylactic vaccine that is potent, safe, and applicable, we constructed an Fc-based form of gp350 to serve as a dimeric antigen. Here, we show that the Fc-based gp350 antigen exhibits dramatically enhanced immunogenicity compared with wild-type gp350 protein. The complete or partial gp350 ectodomain was fused with the mouse IgG2a Fc domain. Fusion with the Fc domain did not impair gp350 folding, binding to a conformation-dependent neutralizing antibody (nAb) and binding to its receptor by enzyme-linked immunosorbent assay and surface plasmon resonance. Specific antibody titers against gp350 were notably enhanced by immunization with gp350-Fc dimers compared with gp350 monomers. Furthermore, immunization with gp350-Fc fusion proteins elicited potent nAbs against EBV. Our data strongly suggest that an EBV gp350 vaccine based on Fc fusion proteins may be an efficient candidate to prevent EBV infection in clinical applications. PMID:29765376

Structure and assembly of the essential RNA ring component of a viral DNA packaging motor

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

Ding, Fang; Lu, Changrui; Zhao, Wei

2011-07-25

Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage {psi}29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 {angstrom} resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNAmore » cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of {psi}29 DNA.« less

Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry.

PubMed

Borrenberghs, Doortje; Dirix, Lieve; De Wit, Flore; Rocha, Susana; Blokken, Jolien; De Houwer, Stéphanie; Gijsbers, Rik; Christ, Frauke; Hofkens, Johan; Hendrix, Jelle; Debyser, Zeger

2016-11-10

Nuclear entry is a selective, dynamic process granting the HIV-1 pre-integration complex (PIC) access to the chromatin. Classical analysis of nuclear entry of heterogeneous viral particles only yields averaged information. We now have employed single-virus fluorescence methods to follow the fate of single viral pre-integration complexes (PICs) during infection by visualizing HIV-1 integrase (IN). Nuclear entry is associated with a reduction in the number of IN molecules in the complexes while the interaction with LEDGF/p75 enhances IN oligomerization in the nucleus. Addition of LEDGINs, small molecule inhibitors of the IN-LEDGF/p75 interaction, during virus production, prematurely stabilizes a higher-order IN multimeric state, resulting in stable IN multimers resistant to a reduction in IN content and defective for nuclear entry. This suggests that a stringent size restriction determines nuclear pore entry. Taken together, this work demonstrates the power of single-virus imaging providing crucial insights in HIV replication and enabling mechanism-of-action studies.

Biogenic manganese oxide nanoparticle formation by a multimeric multicopper oxidase Mnx

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

Romano, Christine A.; Zhou, Mowei; Song, Yang

Bacteria that produce Mn oxides are extraordinarily skilled engineers of nanomaterials that contribute significantly to global biogeochemical cycles. Their enzyme-based reaction mechanisms may be genetically tailored for environmental remediation applications or bioenergy production. However, significant challenges exist for structural characterization of the enzymes responsible for biomineralization. The active Mn oxidase, Mnx, in Bacillus sp. PL-12 is a complex composed of a multicopper oxidase (MCO), MnxG, and two accessory proteins MnxE and MnxF. MnxG shares sequence similarity with other, structurally characterized MCOs. However, MnxE and MnxF have no similarity to any characterized proteins. The ~200 kDa complex has been recalcitrant tomore » crystallization, so its structure is unknown. In this study, native mass spectrometry defines the subunit topology and copper binding of the Mnx complex, while high resolution electron microscopy visualizes the protein and nascent Mn oxide minerals. These data provide critical structural information for conceptualizing how Mnx produces nanoparticulate Mn oxides.« less

Atomic structure of the APC/C and its mechanism of protein ubiquitination

PubMed Central

Yang, Jing; McLaughlin, Stephen H.; Barford, David

2015-01-01

The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex, and interphase inhibitor Emi1 ensures the correct order and timing of distinct cell cycle transitions. Here, we used cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module, and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of experiments to further investigate APC/C functions in vivo. PMID:26083744

Production of in vivo biotinylated scFv specific to almond (Prunus dulcis) proteins by recombinant Pichia pastoris.

PubMed

de la Cruz, Silvia; Alcocer, Marcos; Madrid, Raquel; García, Aina; Martín, Rosario; González, Isabel; García, Teresa

2016-06-10

The methylotropic yeast Pichia pastoris has demonstrated its suitability for large-scale production of recombinant proteins. As an eukaryotic organism P. pastoris presents a series of advantages at expression and processing of heterologous proteins when compared with Escherichia coli. In this work, P. pastoris has been used to express a scFv from a human synthetic library previously shown to bind almond proteins. In order to facilitate purification and post processing manipulations, the scFv was engineered with a C-terminal tag and biotinylated in vivo. After purification, biotinylated scFv were bound to avidin conjugated with HRP producing a multimeric scFv. The multimeric scFv showed to maintain their ability to recognize almond protein when assayed in ELISA, reaching a LOD of 470mgkg(-1). This study describes an easy method to produce large quantities of in vivo biotinylated scFv in P. pastoris. By substituting the enzyme or fluorochromes linked to avidin, it will be possible to generate a diverse number of multimeric scFv as probes to suit different analytical platforms in the detection of almond in food products. Copyright © 2016 Elsevier B.V. All rights reserved.

A disulfide-bond A oxidoreductase-like protein (DsbA-L) regulates adiponectin multimerization

PubMed Central

Liu, Meilian; Zhou, Lijun; Xu, Aimin; Lam, Karen S. L.; Wetzel, Michael D.; Xiang, Ruihua; Zhang, Jingjing; Xin, Xiaoban; Dong, Lily Q.; Liu, Feng

2008-01-01

Impairments in adiponectin multimerization lead to defects in adiponectin secretion and function and are associated with diabetes, yet the underlying mechanisms remain largely unknown. We have identified an adiponectin-interacting protein, previously named GST-kappa, by yeast 2-hybrid screening. The adiponectin-interacting protein contains 2 thioredoxin domains and has very little sequence similarity to other GST isoforms. However, this protein shares high sequence and secondary structure homology to bacterial disulfide-bond A oxidoreductase (DsbA) and is thus renamed DsbA-like protein (DsbA-L). DsbA-L is highly expressed in adipose tissue, and its expression level is negatively correlated with obesity in mice and humans. DsbA-L expression in 3T3-L1 adipocytes is stimulated by the insulin sensitizer rosiglitazone and inhibited by the inflammatory cytokine TNFα. Overexpression of DsbA-L promoted adiponectin multimerization while suppressing DsbA-L expression by RNAi markedly and selectively reduced adiponectin levels and secretion in 3T3-L1 adipocytes. Our results identify DsbA-L as a key regulator for adiponectin biosynthesis and uncover a potential new target for developing therapeutic drugs for the treatment of insulin resistance and its associated metabolic disorders. PMID:19011089

Decoding the non-coding RNAs in Alzheimer's disease.

PubMed

Schonrock, Nicole; Götz, Jürgen

2012-11-01

Non-coding RNAs (ncRNAs) are integral components of biological networks with fundamental roles in regulating gene expression. They can integrate sequence information from the DNA code, epigenetic regulation and functions of multimeric protein complexes to potentially determine the epigenetic status and transcriptional network in any given cell. Humans potentially contain more ncRNAs than any other species, especially in the brain, where they may well play a significant role in human development and cognitive ability. This review discusses their emerging role in Alzheimer's disease (AD), a human pathological condition characterized by the progressive impairment of cognitive functions. We discuss the complexity of the ncRNA world and how this is reflected in the regulation of the amyloid precursor protein and Tau, two proteins with central functions in AD. By understanding this intricate regulatory network, there is hope for a better understanding of disease mechanisms and ultimately developing diagnostic and therapeutic tools.

Cooperative Subunit Refolding of a Light-Harvesting Protein through a Self-Chaperone Mechanism.

PubMed

Laos, Alistair J; Dean, Jacob C; Toa, Zi S D; Wilk, Krystyna E; Scholes, Gregory D; Curmi, Paul M G; Thordarson, Pall

2017-07-10

The fold of a protein is encoded by its amino acid sequence, but how complex multimeric proteins fold and assemble into functional quaternary structures remains unclear. Here we show that two structurally different phycobiliproteins refold and reassemble in a cooperative manner from their unfolded polypeptide subunits, without biological chaperones. Refolding was confirmed by ultrafast broadband transient absorption and two-dimensional electronic spectroscopy to probe internal chromophores as a marker of quaternary structure. Our results demonstrate a cooperative, self-chaperone refolding mechanism, whereby the β-subunits independently refold, thereby templating the folding of the α-subunits, which then chaperone the assembly of the native complex, quantitatively returning all coherences. Our results indicate that subunit self-chaperoning is a robust mechanism for heteromeric protein folding and assembly that could also be applied in self-assembled synthetic hierarchical systems. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Activation Domain of the Bovine Papillomavirus E2 Protein Mediates Association of DNA-Bound Dimers to form DNA Loops

NASA Astrophysics Data System (ADS)

Knight, Jonathan D.; Li, Rong; Botchan, Michael

1991-04-01

The E2 transactivator protein of bovine papillomavirus binds its specific DNA target sequence as a dimer. We have found that E2 dimers, performed in solution independent of DNA, exhibit substantial cooperativity of DNA binding as detected by both nitrocellulose filter retention and footprint analysis techniques. If the binding sites are widely spaced, E2 forms stable DNA loops visible by electron microscopy. When three widely separated binding sites reside on te DNA, E2 condenses the molecule into a bow-tie structure. This implies that each E2 dimer has at least two independent surfaces for multimerization. Two naturally occurring shorter forms of the protein, E2C and D8/E2, which function in vivo as repressors of transcription, do not form such loops. Thus, the looping function of E2 maps to the 161-amino acid activation domain. These results support the looping model of transcription activation by enhancers.

Assessment of Dimeric Metal-Glycan Adducts via Isotopic Labeling and Ion Mobility-Mass Spectrometry.

PubMed

Morrison, Kelsey A; Bendiak, Brad K; Clowers, Brian H

2018-05-25

Adduction of multivalent metal ions to glycans has been shown in recent years to produce altered tandem mass spectra with collision-induced dissociation, electron transfer techniques, and photon-based fragmentation approaches. However, these approaches assume the presence of a well-characterized precursor ion population and do not fully account for the possibility of multimeric species for select glycan-metal complexes. With the use of ion mobility separations prior to mass analysis, doubly charged dimers are not necessarily problematic for tandem MS experiments given that monomer and dimer drift times are sufficiently different. However, multistage mass spectrometric experiments performed on glycans adducted to multivalent metals without mobility separation can yield chimeric fragmentation spectra that are essentially a superposition of the fragments from both the monomeric and dimeric adducts. For homodimeric adducts, where the dimer contains two of the same glycan species, this is less of a concern but if heterodimers can form, there exists the potential for erroneous and misleading fragment ions to appear if a heterodimer containing two different isomers is fragmented along with a targeted monomer. We present an assessment of heterodimer formation between a series of six tetrasaccharides, of which three are isomers, adducted with cobalt(II) and a monodeuterated tetrasaccharide. Using ion mobility separations prior to single-stage and tandem mass analysis, the data shown demonstrate that heterodimeric species can indeed form, and that ion mobility separations are highly necessary prior to using tandem techniques on metal-glycan adducts. Graphical Abstract ᅟ.

Assessment of Dimeric Metal-Glycan Adducts via Isotopic Labeling and Ion Mobility-Mass Spectrometry

NASA Astrophysics Data System (ADS)

Morrison, Kelsey A.; Bendiak, Brad K.; Clowers, Brian H.

2018-05-01

Adduction of multivalent metal ions to glycans has been shown in recent years to produce altered tandem mass spectra with collision-induced dissociation, electron transfer techniques, and photon-based fragmentation approaches. However, these approaches assume the presence of a well-characterized precursor ion population and do not fully account for the possibility of multimeric species for select glycan-metal complexes. With the use of ion mobility separations prior to mass analysis, doubly charged dimers are not necessarily problematic for tandem MS experiments given that monomer and dimer drift times are sufficiently different. However, multistage mass spectrometric experiments performed on glycans adducted to multivalent metals without mobility separation can yield chimeric fragmentation spectra that are essentially a superposition of the fragments from both the monomeric and dimeric adducts. For homodimeric adducts, where the dimer contains two of the same glycan species, this is less of a concern but if heterodimers can form, there exists the potential for erroneous and misleading fragment ions to appear if a heterodimer containing two different isomers is fragmented along with a targeted monomer. We present an assessment of heterodimer formation between a series of six tetrasaccharides, of which three are isomers, adducted with cobalt(II) and a monodeuterated tetrasaccharide. Using ion mobility separations prior to single-stage and tandem mass analysis, the data shown demonstrate that heterodimeric species can indeed form, and that ion mobility separations are highly necessary prior to using tandem techniques on metal-glycan adducts.

Characterization of U(VI)-carbonato ternary complexes on hematite: EXAFS and electrophoretic mobility measurements

USGS Publications Warehouse

Bargar, John R.; Reitmeyer, Rebecca; Lenhart, John J.; Davis, James A.

2000-01-01

We have measured U(VI) adsorption on hematite using EXAFS spectroscopy and electrophoresis under conditions relevant to surface waters and aquifers (0.01 to 10 μM dissolved uranium concentrations, in equilibrium with air, pH 4.5 to 8.5). Both techniques suggest the existence of anionic U(VI)-carbonato ternary complexes. Fits to EXAFS spectra indicate that U(VI) is simultaneously coordinated to surface FeO6 octahedra and carbonate (or bicarbonate) ligands in bidentate fashions, leading to the conclusion that the ternary complexes have an inner-sphere metal bridging (hematite-U(VI)-carbonato) structure. Greater than or equal to 50% of adsorbed U(VI) was comprised of monomeric hematite-U(VI)-carbonato ternary complexes, even at pH 4.5. Multimeric U(VI) species were observed at pH ≥ 6.5 and aqueous U(VI) concentrations approximately an order of magnitude more dilute than the solubility of crystalline β-UO2(OH)2. Based on structural constraints, these complexes were interpreted as dimeric hematite-U(VI)-carbonato ternary complexes. These results suggest that Fe-oxide-U(VI)-carbonato complexes are likely to be important transport-limiting species in oxic aquifers throughout a wide range of pH values.

A simple method for determining polymeric IgA-containing immune complexes.

PubMed

Sancho, J; Egido, J; González, E

1983-06-10

A simplified assay to measure polymeric IgA-immune complexes in biological fluids is described. The assay is based upon the specific binding of a secretory component for polymeric IgA. In the first step, multimeric IgA (monomeric and polymeric) immune complexes are determined by the standard Raji cell assay. Secondly, labeled secretory component added to the assay is bound to polymeric IgA-immune complexes previously fixed to Raji cells, but not to monomeric IgA immune complexes. To avoid false positives due to possible complement-fixing IgM immune complexes, prior IgM immunoadsorption is performed. Using anti-IgM antiserum coupled to CNBr-activated Sepharose 4B this step is not time-consuming. Polymeric IgA has a low affinity constant and binds weakly to Raji cells, as Scatchard analysis of the data shows. Thus, polymeric IgA immune complexes do not bind to Raji cells directly through Fc receptors, but through complement breakdown products, as with IgG-immune complexes. Using this method, we have been successful in detecting specific polymeric-IgA immune complexes in patients with IgA nephropathy (Berger's disease) and alcoholic liver disease, as well as in normal subjects after meals of high protein content. This new, simple, rapid and reproducible assay might help to study the physiopathological role of polymeric IgA immune complexes in humans and animals.

From Molecules to Life: Quantifying the Complexity of Chemical and Biological Systems in the Universe.

PubMed

Böttcher, Thomas

2018-01-01

Life is a complex phenomenon and much research has been devoted to both understanding its origins from prebiotic chemistry and discovering life beyond Earth. Yet, it has remained elusive how to quantify this complexity and how to compare chemical and biological units on one common scale. Here, a mathematical description of molecular complexity was applied allowing to quantitatively assess complexity of chemical structures. This in combination with the orthogonal measure of information complexity resulted in a two-dimensional complexity space ranging over the entire spectrum from molecules to organisms. Entities with a certain level of information complexity directly require a functionally complex mechanism for their production or replication and are hence indicative for life-like systems. In order to describe entities combining molecular and information complexity, the term biogenic unit was introduced. Exemplified biogenic unit complexities were calculated for ribozymes, protein enzymes, multimeric protein complexes, and even an entire virus particle. Complexities of prokaryotic and eukaryotic cells, as well as multicellular organisms, were estimated. Thereby distinct evolutionary stages in complexity space were identified. The here developed approach to compare the complexity of biogenic units allows for the first time to address the gradual characteristics of prebiotic and life-like systems without the need for a definition of life. This operational concept may guide our search for life in the Universe, and it may direct the investigations of prebiotic trajectories that lead towards the evolution of complexity at the origins of life.

Fat-Specific DsbA-L Overexpression Promotes Adiponectin Multimerization and Protects Mice From Diet-Induced Obesity and Insulin Resistance

PubMed Central

Liu, Meilian; Xiang, Ruihua; Wilk, Sarah Ann; Zhang, Ning; Sloane, Lauren B.; Azarnoush, Kian; Zhou, Lijun; Chen, Hongzhi; Xiang, Guangda; Walter, Christi A.; Austad, Steven N.; Musi, Nicolas; DeFronzo, Ralph A.; Asmis, Reto; Scherer, Philipp E.; Dong, Lily Q.; Liu, Feng

2012-01-01

The antidiabetic and antiatherosclerotic effects of adiponectin make it a desirable drug target for the treatment of metabolic and cardiovascular diseases. However, the adiponectin-based drug development approach turns out to be difficult due to extremely high serum levels of this adipokine. On the other hand, a significant correlation between adiponectin multimerization and its insulin-sensitizing effects has been demonstrated, suggesting a promising alternative therapeutic strategy. Here we show that transgenic mice overexpressing disulfide bond A oxidoreductase-like protein in fat (fDsbA-L) exhibited increased levels of total and the high-molecular-weight form of adiponectin compared with wild-type (WT) littermates. The fDsbA-L mice also displayed resistance to diet-induced obesity, insulin resistance, and hepatic steatosis compared with WT control mice. The protective effects of DsbA-L overexpression on diet-induced insulin resistance, but not increased body weight and fat cell size, were significantly decreased in adiponectin-deficient fDsbA-L mice (fDsbA-L/Ad−/−). In addition, the fDsbA-L/Ad−/− mice displayed greater activity and energy expenditure compared with adiponectin knockout mice under a high-fat diet. Taken together, our results demonstrate that DsbA-L protects mice from diet-induced obesity and insulin resistance through adiponectin-dependent and independent mechanisms. In addition, upregulation of DsbA-L could be an effective therapeutic approach for the treatment of obesity and its associated metabolic disorders. PMID:22807031

The roles of protein disulphide isomerase family A, member 3 (ERp57) and surface thiol/disulphide exchange in human spermatozoa-zona pellucida binding.

PubMed

Wong, Chi-Wai; Lam, Kevin K W; Lee, Cheuk-Lun; Yeung, William S B; Zhao, Wei E; Ho, Pak-Chung; Ou, Jian-Ping; Chiu, Philip C N

2017-04-01

Are multimeric sperm plasma membrane protein complexes, ERp57 and sperm surface thiol content involved in human spermatozoa-zona pellucida (ZP) interaction? ERp57 is a component of a multimeric spermatozoa-ZP receptor complex involved in regulation of human spermatozoa-ZP binding via up-regulation of sperm surface thiol content. A spermatozoon acquires its fertilization capacity within the female reproductive tract by capacitation. Spermatozoa-ZP receptor is suggested to be a composite structure that is assembled into a functional complex during capacitation. Sperm surface thiol content is elevated during capacitation. ERp57 is a protein disulphide isomerase that modulates the thiol-disulphide status of proteins. The binding ability and components of protein complexes in extracted membrane protein fractions of spermatozoa were studied. The roles of capacitation, thiol-disulphide reagent treatments and ERp57 on sperm functions and sperm surface thiol content were assessed. Spermatozoa were obtained from semen samples from normozoospermic men. Human oocytes were obtained from an assisted reproduction programme. Blue native polyacrylamide gel electrophoresis, western ligand blotting and mass spectrometry were used to identify the components of solubilized ZP/ZP3-binding complexes. The localization and expression of sperm surface thiol and ERp57 were studied by immunostaining and sperm surface protein biotinylation followed by western blotting. Sperm functions were assessed by standard assays. Several ZP-binding complexes were isolated from the cell membrane of capacitated spermatozoa. ERp57 was a component of one of these complexes. Capacitation significantly increased the sperm surface thiol content, acrosomal thiol distribution and ERp57 expression on sperm surface. Sperm surface thiol and ERp57 immunoreactivity were localized to the acrosomal region of spermatozoa, a region responsible for ZP-binding. Up-regulation of the surface thiol content or ERp57 surface expression in vitro stimulated ZP-binding capacity of human spermatozoa. Blocking of ERp57 function by specific antibody or inhibitors against ERp57 reduced the surface thiol content and ZP-binding capacity of human spermatozoa. N/A. The mechanisms by which up-regulation of surface thiol content stimulates spermatozoa-ZP binding have not been depicted. Thiol-disulphide exchange is a crucial event in capacitation. ERp57 modulates the event and the subsequent fertilization process. Modulation of the surface thiol content of the spermatozoa of subfertile men may help to increase fertilization rate in assisted reproduction. This work was supported by The Hong Kong Research Grant Council Grant HKU764611 and HKU764512M to P.C.N.C. The authors have no competing interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA.

PubMed

Stoltenburg, Regina; Krafčiková, Petra; Víglaský, Viktor; Strehlitz, Beate

2016-09-21

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus, and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5'-end also contributes essentially to the aptameric function.

G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA

PubMed Central

Stoltenburg, Regina; Krafčiková, Petra; Víglaský, Viktor; Strehlitz, Beate

2016-01-01

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus, and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5′-end also contributes essentially to the aptameric function. PMID:27650576

RIP-seq analysis of eukaryotic Sm proteins identifies three major categories of Sm-containing ribonucleoproteins

PubMed Central

2014-01-01

Background Sm proteins are multimeric RNA-binding factors, found in all three domains of life. Eukaryotic Sm proteins, together with their associated RNAs, form small ribonucleoprotein (RNP) complexes important in multiple aspects of gene regulation. Comprehensive knowledge of the RNA components of Sm RNPs is critical for understanding their functions. Results We developed a multi-targeting RNA-immunoprecipitation sequencing (RIP-seq) strategy to reliably identify Sm-associated RNAs from Drosophila ovaries and cultured human cells. Using this method, we discovered three major categories of Sm-associated transcripts: small nuclear (sn)RNAs, small Cajal body (sca)RNAs and mRNAs. Additional RIP-PCR analysis showed both ubiquitous and tissue-specific interactions. We provide evidence that the mRNA-Sm interactions are mediated by snRNPs, and that one of the mechanisms of interaction is via base pairing. Moreover, the Sm-associated mRNAs are mature, indicating a splicing-independent function for Sm RNPs. Conclusions This study represents the first comprehensive analysis of eukaryotic Sm-containing RNPs, and provides a basis for additional functional analyses of Sm proteins and their associated snRNPs outside of the context of pre-mRNA splicing. Our findings expand the repertoire of eukaryotic Sm-containing RNPs and suggest new functions for snRNPs in mRNA metabolism. PMID:24393626

Proteolytic cleavage of polymeric tau protein by caspase-3: implications for Alzheimer disease.

PubMed

Jarero-Basulto, Jose J; Luna-Muñoz, Jose; Mena, Raul; Kristofikova, Zdena; Ripova, Daniela; Perry, George; Binder, Lester I; Garcia-Sierra, Francisco

2013-12-01

Truncated tau protein at Asp(421) is associated with neurofibrillary pathology in Alzheimer disease (AD); however, little is known about its presence in the form of nonfibrillary aggregates. Here, we report immunohistochemical staining of the Tau-C3 antibody, which recognizes Asp(421)-truncated tau, in a group of AD cases with different extents of cognitive impairment. In the hippocampus, we found distinct nonfibrillary aggregates of Asp(421)-truncated tau. Unlike Asp(421)-composed neurofibrillary tangles, however, these nonfibrillary pathologies did not increase significantly with respect to the Braak staging and, therefore, make no significant contribution to cognitive impairment. On the other hand, despite in vitro evidence that caspase-3 cleaves monomeric tau at Asp(421), to date, this truncation has not been demonstrated to be executed by this protease in polymeric tau entities. We determined that Asp(421) truncation can be produced by caspase-3 in oligomeric and multimeric complexes of recombinant full-length tau in isolated native tau filaments in vitro and in situ in neurofibrillary tangles analyzed in fresh brain slices from AD cases. Our data suggest that generation of this pathologic Asp(421) truncation of tau in long-lasting fibrillary structures may produce further permanent toxicity for neurons in the brains of patients with AD.

InterEvDock2: an expanded server for protein docking using evolutionary and biological information from homology models and multimeric inputs.

PubMed

Quignot, Chloé; Rey, Julien; Yu, Jinchao; Tufféry, Pierre; Guerois, Raphaël; Andreani, Jessica

2018-05-08

Computational protein docking is a powerful strategy to predict structures of protein-protein interactions and provides crucial insights for the functional characterization of macromolecular cross-talks. We previously developed InterEvDock, a server for ab initio protein docking based on rigid-body sampling followed by consensus scoring using physics-based and statistical potentials, including the InterEvScore function specifically developed to incorporate co-evolutionary information in docking. InterEvDock2 is a major evolution of InterEvDock which allows users to submit input sequences - not only structures - and multimeric inputs and to specify constraints for the pairwise docking process based on previous knowledge about the interaction. For this purpose, we added modules in InterEvDock2 for automatic template search and comparative modeling of the input proteins. The InterEvDock2 pipeline was benchmarked on 812 complexes for which unbound homology models of the two partners and co-evolutionary information are available in the PPI4DOCK database. InterEvDock2 identified a correct model among the top 10 consensus in 29% of these cases (compared to 15-24% for individual scoring functions) and at least one correct interface residue among 10 predicted in 91% of these cases. InterEvDock2 is thus a unique protein docking server, designed to be useful for the experimental biology community. The InterEvDock2 web interface is available at http://bioserv.rpbs.univ-paris-diderot.fr/services/InterEvDock2/.

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins.

PubMed

Ramsey, Kathryn A; Rushton, Zachary L; Ehre, Camille

2016-06-14

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS).

PubMed

Lou, Tzu-Fang; Weidmann, Chase A; Killingsworth, Jordan; Tanaka Hall, Traci M; Goldstrohm, Aaron C; Campbell, Zachary T

2017-04-15

RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA. Copyright © 2016 Elsevier Inc. All rights reserved.

A TBP-containing multiprotein complex (TIF-IB) mediates transcription specificity of murine RNA polymerase I.

PubMed

Eberhard, D; Tora, L; Egly, J M; Grummt, I

1993-09-11

TIF-IB is a transcription factor which interacts with the mouse ribosomal gene promoter and nucleates the formation of an initiation complex containing RNA polymerase I (Pol I). We have purified this factor to near homogeneity and demonstrate that TIF-IB is a large complex (< 200 kDa) which contains several polypeptides. One of the subunits present in this protein complex is the TATA-binding protein (TBP) as revealed by copurification of TIF-IB activity and TBP over different chromatographic steps including immunoaffinity purification. In addition to TBP, three tightly associated proteins (TAFs-I) with apparent molecular weights of 95, 68, and 48 kDa are contained in this multimeric complex. This subunit composition is similar--but not identical--to the analogous human factor SL1. Depletion of TBP from TIF-IB-containing fractions by immunoprecipitation eliminates TIF-IB activity. Neither TBP alone nor fractions containing other TBP complexes are capable of substituting for TIF-IB activity. Therefore, TIF-IB is a unique complex with Pol I-specific TAFs distinct from other TBP-containing complexes. The identification of TBP as an integral part of the murine rDNA promoter-specific transcription initiation factor extends the previously noted similarity of transcriptional initiation by the three nuclear RNA polymerases and underscores the importance of TAFs in determining promoter specificity.

A TBP-containing multiprotein complex (TIF-IB) mediates transcription specificity of murine RNA polymerase I.

PubMed Central

Eberhard, D; Tora, L; Egly, J M; Grummt, I

1993-01-01

TIF-IB is a transcription factor which interacts with the mouse ribosomal gene promoter and nucleates the formation of an initiation complex containing RNA polymerase I (Pol I). We have purified this factor to near homogeneity and demonstrate that TIF-IB is a large complex (< 200 kDa) which contains several polypeptides. One of the subunits present in this protein complex is the TATA-binding protein (TBP) as revealed by copurification of TIF-IB activity and TBP over different chromatographic steps including immunoaffinity purification. In addition to TBP, three tightly associated proteins (TAFs-I) with apparent molecular weights of 95, 68, and 48 kDa are contained in this multimeric complex. This subunit composition is similar--but not identical--to the analogous human factor SL1. Depletion of TBP from TIF-IB-containing fractions by immunoprecipitation eliminates TIF-IB activity. Neither TBP alone nor fractions containing other TBP complexes are capable of substituting for TIF-IB activity. Therefore, TIF-IB is a unique complex with Pol I-specific TAFs distinct from other TBP-containing complexes. The identification of TBP as an integral part of the murine rDNA promoter-specific transcription initiation factor extends the previously noted similarity of transcriptional initiation by the three nuclear RNA polymerases and underscores the importance of TAFs in determining promoter specificity. Images PMID:8414971

Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton

PubMed Central

Takeda, Tetsuro; McQuistan, Tammie; Orlando, Robert A.; Farquhar, Marilyn G.

2001-01-01

Podocalyxin (PC), the major sialoprotein of glomerular epithelial cells (GECs), helps maintain the characteristic architecture of the foot processes and the patency of the filtration slits. PC associates with actin via ezrin, a member of the ERM family of cytoskeletal linker proteins. Here we show that PC is linked to ezrin and the actin cytoskeleton via Na+/H+-exchanger regulatory factor 2 (NHERF2), a scaffold protein containing two PDZ (PSD-95/Dlg/ZO-1) domains and an ERM-binding region. The cytoplasmic tail of PC contains a C-terminal PDZ-binding motif (DTHL) that binds to the second PDZ domain of NHERF2 in yeast two-hybrid and in vitro pull-down assays. By immunocytochemistry NHERF2 colocalizes with PC and ezrin along the apical domain of the GEC plasma membrane. NHERF2 and ezrin form a multimeric complex with PC, as they coimmunoprecipitate with PC. The PC/NHERF2/ezrin complex interacts with the actin cytoskeleton, and this interaction is disrupted in GECs from puromycin aminonucleoside–, protamine sulfate–, or sialidase-treated rats, which show a dramatic loss of foot processes, comparable to that seen in the nephrotic syndrome. Thus NHERF2 appears to function as a scaffold protein linking PC to ezrin and the actin cytoskeleton. PC/NHERF2/ezrin/actin interactions are disrupted in pathologic conditions associated with changes in GEC foot processes, indicating their importance for maintaining the unique organization of this epithelium. J. Clin. Invest. 108:289–301 (2001). DOI:10.1172/JCI200112539. PMID:11457882

Stepwise assembly of multiple Lin28 proteins on the terminal loop of let-7 miRNA precursors

PubMed Central

Desjardins, Alexandre; Bouvette, Jonathan; Legault, Pascale

2014-01-01

Lin28 inhibits the biogenesis of let-7 miRNAs through direct interactions with let-7 precursors. Previous studies have described seemingly inconsistent Lin28 binding sites on pre-let-7 RNAs. Here, we reconcile these data by examining the binding mechanism of Lin28 to the terminal loop of pre-let-7g (TL-let-7g) using biochemical and biophysical methods. First, we investigate Lin28 binding to TL-let-7g variants and short RNA fragments and identify three independent binding sites for Lin28 on TL-let-7g. We then determine that Lin28 assembles in a stepwise manner on TL-let-7g to form a stable 1:3 complex. We show that the cold-shock domain (CSD) of Lin28 is responsible for remodelling the terminal loop of TL-let-7g, whereas the NCp7-like domain facilitates the initial binding of Lin28 to TL-let-7g. This stable binding of multiple Lin28 molecules to the terminal loop of pre-let-7g extends to other precursors of the let-7 family, but not to other pre-miRNAs tested. We propose a model for stepwise assembly of the 1:1, 1:2 and 1:3 pre-let-7g/Lin28 complexes. Stepwise multimerization of Lin28 on pre-let-7 is required for maximum inhibition of Dicer cleavage for a least one member of the let-7 family and may be important for orchestrating the activity of the several factors that regulate let-7 biogenesis. PMID:24452802

Outer membrane components of the Tad (tight adherence) secreton of Aggregatibacter actinomycetemcomitans.

PubMed

Clock, Sarah A; Planet, Paul J; Perez, Brenda A; Figurski, David H

2008-02-01

Prokaryotic secretion relies on proteins that are widely conserved, including NTPases and secretins, and on proteins that are system specific. The Tad secretion system in Aggregatibacter actinomycetemcomitans is dedicated to the assembly and export of Flp pili, which are needed for tight adherence. Consistent with predictions that RcpA forms the multimeric outer membrane secretion channel (secretin) of the Flp pilus biogenesis apparatus, we observed the RcpA protein in multimers that were stable in the presence of detergent and found that rcpA and its closely related homologs form a novel and distinct subfamily within a well-supported gene phylogeny of the entire secretin gene superfamily. We also found that rcpA-like genes were always linked to Aggregatibacter rcpB- or Caulobacter cpaD-like genes. Using antisera, we determined the localization and gross abundances of conserved (RcpA and TadC) and unique (RcpB, RcpC, and TadD) Tad proteins. The three Rcp proteins (RcpA, RcpB, and RcpC) and TadD, a putative lipoprotein, localized to the bacterial outer membrane. RcpA, RcpC, and TadD were also found in the inner membrane, while TadC localized exclusively to the inner membrane. The RcpA secretin was necessary for wild-type abundances of RcpB and RcpC, and TadC was required for normal levels of all three Rcp proteins. TadC abundance defects were observed in rcpA and rcpC mutants. TadD production was essential for wild-type RcpA and RcpB abundances, and RcpA did not multimerize or localize to the outer membrane without the expression of TadD. These data indicate that membrane proteins TadC and TadD may influence the assembly, transport, and/or function of individual outer membrane Rcp proteins.

A unique mid-sequence linker used to multimerize the lipid-phosphatidylserine (PS) binding peptide-peptoid hybrid PPS1.

PubMed

Shukla, Satya Prakash; Manarang, Joseph C; Udugamasooriya, D Gomika

2017-09-08

Ligand multimerizations enhance the binding affinity towards cell surface biomarkers through their avidity effects. Typical linkers connect individual monomeric ligand moieties from one end (e.g., C- or N-terminus of a peptide) and exclusively target protein receptors. The lipid phosphatidylserine (PS) is normally present on the cytoplasmic side of the eukaryotic cell membrane, but in tumors and tumor endothelial cells, this negatively charged PS flips to the outer layer. We recently reported a PS binding peptide-peptoid hybrid (PPS1) that has distinct positively charged and hydrophobic residue-containing regions. The PPS1 monomer is inactive, and upon C-terminal dimerization (PPS1D1), it triggers cytotoxicity. In the current study, a unique series of PPS1 multimeric derivatives were synthesized by switching the linker from the C-terminus to an internal position. The unimportant fourth residue (N-lys) from the C-terminus was utilized to build the linker. The synthesis strategy was developed employing variations of (I) the linker size, (II) the number of positively charged residues, and (III) the number of hydrophobic regions. Cytotoxicity of these new derivatives on HCC4017 lung cancer cells showed that a minimum of two hydrophobic regions was important to retain the activity and that the shortest linker length was optimal for activity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Dimeric MHC-peptides inserted into an immunoglobulin scaffold as new immunotherapeutic agents

PubMed Central

Goldberg, Burt; Bona, Constantin

2011-01-01

Abstract The interactions of the T cell receptor (TCR) with cognate MHC-peptide and co-stimulatory molecules expressed at surface of antigen presenting cells (APC) leads to activation or tolerance of T cells. The development of molecular biological tools allowed for the preparation of soluble MHC-peptide molecules as surrogate for the APC. A decade ago a monomeric class II MHC molecule in which the peptide was covalently linked to β-chain of class II molecule was generated. This type of molecule had a low-binding affinity and did not cause the multimerization of TCR. The requirement of multimerization of TCR led to development of a new class of reagents, chimeric peptides covalently linked to MHC that was dimerized via Fc fragment of an immunoglobulin and linked to 3′ end of the β-chain of MHC class II molecule. These soluble dimerized MHC-peptide chimeric molecules display high affinity for the TCR and caused multimerization of TCR without processing by an APC. Because dimeric molecules are devoid of co-stimulatory molecules interacting with CD28, a second signal, they induce anergy rather the activation of T cells. In this review, we compare the human and murine dimerized MHC class II-peptides and their effect on CD4+ T cells, particularly the generation of T regulatory cells, which make these chimeric molecules an appealing approach for the treatment of autoimmune diseases. PMID:21435177

A general path for large-scale solubilization of cellular proteins: From membrane receptors to multiprotein complexes

PubMed Central

Pullara, Filippo; Guerrero-Santoro, Jennifer; Calero, Monica; Zhang, Qiangmin; Peng, Ye; Spåhr, Henrik; Kornberg, Guy L.; Cusimano, Antonella; Stevenson, Hilary P.; Santamaria-Suarez, Hugo; Reynolds, Shelley L.; Brown, Ian S.; Monga, Satdarshan P.S.; Van Houten, Bennett; Rapić-Otrin, Vesna; Calero, Guillermo; Levine, Arthur S.

2014-01-01

Expression of recombinant proteins in bacterial or eukaryotic systems often results in aggregation rendering them unavailable for biochemical or structural studies. Protein aggregation is a costly problem for biomedical research. It forces research laboratories and the biomedical industry to search for alternative, more soluble, non-human proteins and limits the number of potential “druggable” targets. In this study we present a highly reproducible protocol that introduces the systematic use of an extensive number of detergents to solubilize aggregated proteins expressed in bacterial and eukaryotic systems. We validate the usefulness of this protocol by solubilizing traditionally difficult human protein targets to milligram quantities and confirm their biological activity. We use this method to solubilize monomeric or multimeric components of multi-protein complexes and demonstrate its efficacy to reconstitute large cellular machines. This protocol works equally well on cytosolic, nuclear and membrane proteins and can be easily adapted to a high throughput format. PMID:23137940

Insights into MHC class I peptide loading from the structure of the tapasin/ERp57 heterodimer

PubMed Central

Dong, Gang; Wearsch, Pamela A.; Peaper, David R.; Cresswell, Peter; Reinisch, Karin M.

2009-01-01

SUMMARY Tapasin is a glycoprotein critical for loading Major Histocompatibility Complex (MHC) class I molecules with high affinity peptides. It functions within the multimeric peptide-loading complex (PLC) as a disulfide-linked, stable heterodimer with the thiol oxidoreductase ERp57, and this covalent interaction is required to support optimal PLC activity. Here we present the 2.6 Å resolution structure of the tapasin/ERp57 core of the PLC. The structure reveals the basis for the stable dimerization of tapasin and ERp57 and provides the first example of a protein disulfide isomerase family member interacting with a substrate. Mutational analysis identified a conserved surface on tapasin that interacts with MHC class I molecules and is critical for the peptide loading and editing function of the tapasin-ERp57 heterodimer. By combining the tapasin/ERp57 structure with those of other defined PLC components we present a molecular model that illuminates the processes involved in MHC class I peptide loading. PMID:19119025

Molecular basis of APC/C regulation by the spindle assembly checkpoint

PubMed Central

Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

2016-01-01

In the dividing eukaryotic cell the spindle assembly checkpoint (SAC) ensures each daughter cell inherits an identical set of chromosomes. The SAC coordinates the correct attachment of sister chromatid kinetochores to the mitotic spindle with activation of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome separation. In response to unattached kinetochores, the SAC generates the mitotic checkpoint complex (MCC), a multimeric assembly that inhibits the APC/C, delaying chromosome segregation. Here, using cryo-electron microscopy we determined the near-atomic resolution structure of an APC/C-MCC complex (APC/CMCC). We reveal how degron-like sequences of the MCC subunit BubR1 block degron recognition sites on Cdc20, the APC/C coactivator subunit (Cdc20APC/C) responsible for substrate interactions. BubR1 also obstructs binding of UbcH10 (APC/C’s initiating E2) to repress APC/C ubiquitination activity. Conformational variability of the complex allows for UbcH10 association, and we show from a structure of APC/CMCC in complex with UbcH10 how the Cdc20 subunit intrinsic to the MCC (Cdc20MCC) is ubiquitinated, a process that results in APC/C reactivation when the SAC is silenced. PMID:27509861

Roles of Residues Arg-61 and Gln-38 of Human DNA Polymerase η in Bypass of Deoxyguanosine and 7,8-Dihydro-8-oxo-2'-deoxyguanosine.

PubMed

Su, Yan; Patra, Amritraj; Harp, Joel M; Egli, Martin; Guengerich, F Peter

2015-06-26

Like the other Y-family DNA polymerases, human DNA polymerase η (hpol η) has relatively low fidelity and is able to tolerate damage during DNA synthesis, including 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG), one of the most abundant DNA lesions in the genome. Crystal structures show that Arg-61 and Gln-38 are located near the active site and may play important roles in the fidelity and efficiency of hpol η. Site-directed mutagenesis was used to replace these side chains either alone or together, and the wild type or mutant proteins were purified and tested by replicating DNA past deoxyguanosine (G) or 8-oxoG. The catalytic activity of hpol η was dramatically disrupted by the R61M and Q38A/R61A mutations, as opposed to the R61A and Q38A single mutants. Crystal structures of hpol η mutant ternary complexes reveal that polarized water molecules can mimic and partially compensate for the missing side chains of Arg-61 and Gln-38 in the Q38A/R61A mutant. The combined data indicate that the positioning and positive charge of Arg-61 synergistically contribute to the nucleotidyl transfer reaction, with additional influence exerted by Gln-38. In addition, gel filtration chromatography separated multimeric and monomeric forms of wild type and mutant hpol η, indicating the possibility that hpol η forms multimers in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Roles of Residues Arg-61 and Gln-38 of Human DNA Polymerase η in Bypass of Deoxyguanosine and 7,8-Dihydro-8-oxo-2′-deoxyguanosine*

PubMed Central

Su, Yan; Patra, Amritraj; Harp, Joel M.; Egli, Martin; Guengerich, F. Peter

2015-01-01

Like the other Y-family DNA polymerases, human DNA polymerase η (hpol η) has relatively low fidelity and is able to tolerate damage during DNA synthesis, including 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG), one of the most abundant DNA lesions in the genome. Crystal structures show that Arg-61 and Gln-38 are located near the active site and may play important roles in the fidelity and efficiency of hpol η. Site-directed mutagenesis was used to replace these side chains either alone or together, and the wild type or mutant proteins were purified and tested by replicating DNA past deoxyguanosine (G) or 8-oxoG. The catalytic activity of hpol η was dramatically disrupted by the R61M and Q38A/R61A mutations, as opposed to the R61A and Q38A single mutants. Crystal structures of hpol η mutant ternary complexes reveal that polarized water molecules can mimic and partially compensate for the missing side chains of Arg-61 and Gln-38 in the Q38A/R61A mutant. The combined data indicate that the positioning and positive charge of Arg-61 synergistically contribute to the nucleotidyl transfer reaction, with additional influence exerted by Gln-38. In addition, gel filtration chromatography separated multimeric and monomeric forms of wild type and mutant hpol η, indicating the possibility that hpol η forms multimers in vivo. PMID:25947374

Essential multimeric enzymes in kinetoplastid parasites: A host of potentially druggable protein-protein interactions.

PubMed

Wachsmuth, Leah M; Johnson, Meredith G; Gavenonis, Jason

2017-06-01

Parasitic diseases caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania are an urgent public health crisis in the developing world. These closely related species possess a number of multimeric enzymes in highly conserved pathways involved in vital functions, such as redox homeostasis and nucleotide synthesis. Computational alanine scanning of these protein-protein interfaces has revealed a host of potentially ligandable sites on several established and emerging anti-parasitic drug targets. Analysis of interfaces with multiple clustered hotspots has suggested several potentially inhibitable protein-protein interactions that may have been overlooked by previous large-scale analyses focusing solely on secondary structure. These protein-protein interactions provide a promising lead for the development of new peptide and macrocycle inhibitors of these enzymes.

Myosin light chains: Teaching old dogs new tricks

PubMed Central

Heissler, Sarah M; Sellers, James R

2014-01-01

The myosin holoenzyme is a multimeric protein complex consisting of heavy chains and light chains. Myosin light chains are calmodulin family members which are crucially involved in the mechanoenzymatic function of the myosin holoenzyme. This review examines the diversity of light chains within the myosin superfamily, discusses interactions between the light chain and the myosin heavy chain as well as regulatory and structural functions of the light chain as a subunit of the myosin holoenzyme. It covers aspects of the myosin light chain in the localization of the myosin holoenzyme, protein-protein interactions and light chain binding to non-myosin binding partners. Finally, this review challenges the dogma that myosin regulatory and essential light chain exclusively associate with conventional myosin heavy chains while unconventional myosin heavy chains usually associate with calmodulin. PMID:26155737

Cracks in the beta-can: fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria).

PubMed

Wiedenmann, J; Elke, C; Spindler, K D; Funke, W

2000-12-19

We characterize two green fluorescent proteins (GFPs), an orange fluorescent protein, and a nonfluorescent red protein isolated from the sea anemone Anemonia sulcata. The orange fluorescent protein and the red protein seem to represent two different states of the same protein. Furthermore, we describe the cloning of a GFP and a nonfluorescent red protein. Both proteins are homologous to the GFP from Aequorea victoria. The red protein is significantly smaller than other GFP homologues, and the formation of a closed GFP-like beta-can is not possible. Nevertheless, the primary structure of the red protein carries all features necessary for orange fluorescence. We discuss a type of beta-can that could be formed in a multimerization process.

Cracks in the β-can: Fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria)

PubMed Central

Wiedenmann, Jörg; Elke, Carsten; Spindler, Klaus-Dieter; Funke, Werner

2000-01-01

We characterize two green fluorescent proteins (GFPs), an orange fluorescent protein, and a nonfluorescent red protein isolated from the sea anemone Anemonia sulcata. The orange fluorescent protein and the red protein seem to represent two different states of the same protein. Furthermore, we describe the cloning of a GFP and a nonfluorescent red protein. Both proteins are homologous to the GFP from Aequorea victoria. The red protein is significantly smaller than other GFP homologues, and the formation of a closed GFP-like β-can is not possible. Nevertheless, the primary structure of the red protein carries all features necessary for orange fluorescence. We discuss a type of β-can that could be formed in a multimerization process. PMID:11121018

Resistance to pyridine-based inhibitor KF116 reveals an unexpected role of integrase in HIV-1 Gag-Pol polyprotein proteolytic processing.

PubMed

Hoyte, Ashley C; Jamin, Augusta V; Koneru, Pratibha C; Kobe, Matthew J; Larue, Ross C; Fuchs, James R; Engelman, Alan N; Kvaratskhelia, Mamuka

2017-12-01

The pyridine-based multimerization selective HIV-1 integrase (IN) inhibitors (MINIs) are a distinct subclass of allosteric IN inhibitors. MINIs potently inhibit HIV-1 replication during virion maturation by inducing hyper- or aberrant IN multimerization but are largely ineffective during the early steps of viral replication. Here, we investigated the mechanism for the evolution of a triple IN substitution (T124N/V165I/T174I) that emerges in cell culture with a representative MINI, KF116. We show that HIV-1 NL4-3(IN T124N/V165I/T174I) confers marked (>2000-fold) resistance to KF116. Two IN substitutions (T124N/T174I) directly weaken inhibitor binding at the dimer interface of the catalytic core domain but at the same time markedly impair HIV-1 replication capacity. Unexpectedly, T124N/T174I IN substitutions inhibited proteolytic processing of HIV-1 polyproteins Gag and Gag-Pol, resulting in immature virions. Strikingly, the addition of the third IN substitution (V165I) restored polyprotein processing, virus particle maturation, and significant levels of replication capacity. These results reveal an unanticipated role of IN for polyprotein proteolytic processing during virion morphogenesis. The complex evolutionary pathway for the emergence of resistant viruses, which includes the need for the compensatory V165I IN substitution, highlights a relatively high genetic barrier exerted by MINI KF116. Additionally, we have solved the X-ray structure of the drug-resistant catalytic core domain protein, which provides means for rational development of second-generation MINIs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Lipid-protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins.

PubMed

Shenkarev, Zakhar O; Lyukmanova, Ekaterina N; Butenko, Ivan O; Petrovskaya, Lada E; Paramonov, Alexander S; Shulepko, Mikhail A; Nekrasova, Oksana V; Kirpichnikov, Mikhail P; Arseniev, Alexander S

2013-02-01

Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs. Copyright © 2012 Elsevier B.V. All rights reserved.

Mapping of the self-interaction domains in the simian immunodeficiency virus Gag polyprotein.

PubMed

Rauddi, María L; Mac Donald, Cecilia L; Affranchino, José L; González, Silvia A

2011-03-01

To gain a better understanding of the assembly process in simian immunodeficiency virus (SIV), we first established the conditions under which recombinant SIV Gag lacking the C-terminal p6 domain (SIV GagΔp6) assembled in vitro into spherical particles. Based on the full multimerization capacity of SIV GagΔp6, and to identify the Gag sequences involved in homotypic interactions, we next developed a pull-down assay in which a panel of histidine-tagged SIV Gag truncation mutants was tested for its ability to associate in vitro with GST-SIVGagΔp6. Removal of the nucleocapsid (NC) domain from Gag impaired its ability to interact with GST-SIVGagΔp6. However, this Gag mutant consisting of the matrix (MA) and capsid (CA) domains still retained 50% of the wild-type binding activity. Truncation of SIV Gag from its N-terminus yielded markedly different results. The Gag region consisting of the CA and NC was significantly more efficient than wild-type Gag at interacting in vitro with GST-SIVGagΔp6. Notably, a small Gag subdomain containing the C-terminal third of the CA and the entire NC not only bound to GST-SIVGagΔp6 in vitro at wild-type levels, but also associated in vivo with full-length Gag and was recruited into extracellular particles. Interestingly, when the mature Gag products were analyzed, the MA and NC interacted with GST-SIVGagΔp6 with efficiencies representing 20% and 40%, respectively, of the wild-type value, whereas the CA failed to bind to GST-SIVGagΔp6, despite being capable of self-associating into multimeric complexes.

A rapid method to visualize von willebrand factor multimers by using agarose gel electrophoresis, immunolocalization and luminographic detection.

PubMed

Krizek, D R; Rick, M E

2000-03-15

A highly sensitive and rapid clinical method for the visualization of the multimeric structure of von Willebrand Factor in plasma and platelets is described. The method utilizes submerged horizontal agarose gel electrophoresis, followed by transfer of the von Willebrand Factor onto a polyvinylidine fluoride membrane, and immunolocalization and luminographic visualization of the von Willebrand Factor multimeric pattern. This method distinguishes type 1 from types 2A and 2B von Willebrand disease, allowing timely evaluation and classification of von Willebrand Factor in patient plasma. It also allows visualization of the unusually high molecular weight multimers present in platelets. There are several major advantages to this method including rapid processing, simplicity of gel preparation, high sensitivity to low concentrations of von Willebrand Factor, and elimination of radioactivity.

Probability distributions for multimeric systems.

PubMed

Albert, Jaroslav; Rooman, Marianne

2016-01-01

We propose a fast and accurate method of obtaining the equilibrium mono-modal joint probability distributions for multimeric systems. The method necessitates only two assumptions: the copy number of all species of molecule may be treated as continuous; and, the probability density functions (pdf) are well-approximated by multivariate skew normal distributions (MSND). Starting from the master equation, we convert the problem into a set of equations for the statistical moments which are then expressed in terms of the parameters intrinsic to the MSND. Using an optimization package on Mathematica, we minimize a Euclidian distance function comprising of a sum of the squared difference between the left and the right hand sides of these equations. Comparison of results obtained via our method with those rendered by the Gillespie algorithm demonstrates our method to be highly accurate as well as efficient.

A genetic screen for terminator function in yeast identifies a role for a new functional domain in termination factor Nab3.

PubMed

Loya, Travis J; O'Rourke, Thomas W; Reines, Daniel

2012-08-01

The yeast IMD2 gene encodes an enzyme involved in GTP synthesis. Its expression is controlled by guanine nucleotides through a set of alternate start sites and an intervening transcriptional terminator. In the off state, transcription results in a short non-coding RNA that starts upstream of the gene. Transcription terminates via the Nrd1-Nab3-Sen1 complex and is degraded by the nuclear exosome. Using a sensitive terminator read-through assay, we identified trans-acting Terminator Override (TOV) genes that operate this terminator. Four genes were identified: the RNA polymerase II phosphatase SSU72, the RNA polymerase II binding protein PCF11, the TRAMP subunit TRF4 and the hnRNP-like, NAB3. The TOV phenotype can be explained by the loss of function of these gene products as described in models in which termination and RNA degradation are coupled to the phosphorylation state of RNA polymerase II's repeat domain. The most interesting mutations were those found in NAB3, which led to the finding that the removal of merely three carboxy-terminal amino acids compromised Nab3's function. This region of previously unknown function is distant from the protein's well-known RNA binding and Nrd1 binding domains. Structural homology modeling suggests this Nab3 'tail' forms an α-helical multimerization domain that helps assemble it onto an RNA substrate.

Intraspecific variation in mitochondrial genome sequence, structure, and gene content in Silene vulgaris, an angiosperm with pervasive cytoplasmic male sterility.

PubMed

Sloan, Daniel B; Müller, Karel; McCauley, David E; Taylor, Douglas R; Storchová, Helena

2012-12-01

In angiosperms, mitochondrial-encoded genes can cause cytoplasmic male sterility (CMS), resulting in the coexistence of female and hermaphroditic individuals (gynodioecy). We compared four complete mitochondrial genomes from the gynodioecious species Silene vulgaris and found unprecedented amounts of intraspecific diversity for plant mitochondrial DNA (mtDNA). Remarkably, only about half of overall sequence content is shared between any pair of genomes. The four mtDNAs range in size from 361 to 429 kb and differ in gene complement, with rpl5 and rps13 being intact in some genomes but absent or pseudogenized in others. The genomes exhibit essentially no conservation of synteny and are highly repetitive, with evidence of reciprocal recombination occurring even across short repeats (< 250 bp). Some mitochondrial genes exhibit atypically high degrees of nucleotide polymorphism, while others are invariant. The genomes also contain a variable number of small autonomously mapping chromosomes, which have only recently been identified in angiosperm mtDNA. Southern blot analysis of one of these chromosomes indicated a complex in vivo structure consisting of both monomeric circles and multimeric forms. We conclude that S. vulgaris harbors an unusually large degree of variation in mtDNA sequence and structure and discuss the extent to which this variation might be related to CMS. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Recombinant von Willebrand factor: preclinical development.

PubMed

Plaimauer, B; Schlokat, U; Turecek, P L; Mitterer, A; Mundt, W; Auer, W; Pichler, L; Gritsch, H; Schwarz, H P

2001-08-01

Von Willebrand factor (vWF) is a multimeric glycoprotein (GP) that attracts platelets to the site of vascular injury, mediates platelet-platelet interaction, and stabilizes factor VIII (FVIII) in the circulation. Quantitative and qualitative defects of vWF result in von Willebrand disease (vWD), manifested by modest to severe bleeding episodes. Substitution therapy, with plasma-derived FVIII/vWF complex concentrates, is used for patients suffering the more severe forms of vWD. Efficacy of these preparations is often unsatisfactory because inadvertent proteolytic degradation during the manufacturing process causes them to lack the hemostatically most active high-molecular-weight multimers. In contrast, recombinant vWF (r-vWF), which is constitutively expressed at high yields in Chinese hamster ovary (CHO) cells and secreted into the conditioned medium under perfusion fermentation in "protein-free" medium, has high-molecular-weight multimers of extraordinary structural integrity. Functional analysis has shown that r-vWF promotes ristocetin cofactor-mediated platelet aggregation, collagen interaction and FVIII binding, and platelet-collagen adhesion under shear stress. Infusing vWF-deficient animals with r-vWF corrected vWF concentration and reduced blood loss, subsequently stabilizing endogenous FVIII associated with the reduction of bleeding time. Compared with plasma-derived vWF preparations, r-vWF was found to have a prolonged half-life, further enhancing the potential value of r-vWF as a therapeutic agent for treating patients suffering from vWD.

Functional Analysis of the Anti-adalimumab Response Using Patient-derived Monoclonal Antibodies♦

PubMed Central

van Schouwenburg, Pauline A.; Kruithof, Simone; Votsmeier, Christian; van Schie, Karin; Hart, Margreet H.; de Jong, Rob N.; van Buren, Esther E. L.; van Ham, Marieke; Aarden, Lucien; Wolbink, Gertjan; Wouters, Diana; Rispens, Theo

2014-01-01

The production of antibodies to adalimumab in autoimmune patients treated with adalimumab is shown to diminish treatment efficacy. We previously showed that these antibodies are almost exclusively neutralizing, indicating a restricted response. Here, we investigated the characteristics of a panel of patient-derived monoclonal antibodies for binding to adalimumab. Single B-cells were isolated from two patients, cultured, and screened for adalimumab specificity. Analysis of variable region sequences of 16 clones suggests that the immune response against adalimumab is broad, involving multiple B-cell clones each using different combinations of V(D)J segments. A strong bias for replacement mutations in the complementarity determining regions was found, indicating an antigen-driven response. We recombinantly expressed 11 different monoclonal antibodies and investigated their affinity and specificity. All clones except one are of high affinity (Kd between 0.6 and 233 pm) and compete with TNF as well as each other for binding to adalimumab. However, binding to a panel of single-point mutants of adalimumab indicates markedly different fine specificities that also result in a differential tendency of each clone to form dimeric and multimeric immune complexes. We conclude that although all anti-adalimumab antibodies compete for binding to TNF, the response is clonally diverse and involves multiple epitopes on adalimumab. These results are important for understanding the relationship between self and non-self or idiotypic determinants on therapeutic antibodies and their potential immunogenicity. PMID:25326381

Biogenic manganese oxide nanoparticle formation by a multimeric multicopper oxidase Mnx.

PubMed

Romano, Christine A; Zhou, Mowei; Song, Yang; Wysocki, Vicki H; Dohnalkova, Alice C; Kovarik, Libor; Paša-Tolić, Ljiljana; Tebo, Bradley M

2017-09-29

Bacteria that produce Mn oxides are extraordinarily skilled engineers of nanomaterials that contribute significantly to global biogeochemical cycles. Their enzyme-based reaction mechanisms may be genetically tailored for environmental remediation applications or bioenergy production. However, significant challenges exist for structural characterization of the enzymes responsible for biomineralization. The active Mn oxidase in Bacillus sp. PL-12, Mnx, is a complex composed of a multicopper oxidase (MCO), MnxG, and two accessory proteins, MnxE and MnxF. MnxG shares sequence similarity with other, structurally characterized MCOs. MnxE and MnxF have no similarity to any characterized proteins. The ~200 kDa complex has been recalcitrant to crystallization, so its structure is unknown. Here, we show that native mass spectrometry defines the subunit topology and copper binding of Mnx, while high-resolution electron microscopy visualizes the protein and nascent Mn oxide minerals. These data provide critical structural information for understanding Mn biomineralization by such unexplored enzymes.Significant challenges exist for structural characterization of enzymes responsible for biomineralization. Here the authors show that native mass spectrometry and high resolution electron microscopy can define the subunit topology and copper binding of a manganese oxidizing complex, and describe early stage formation of its mineral products.

Tricomponent Immunopotentiating System as a Novel Molecular Design Strategy for Malaria Vaccine Development ▿

PubMed Central

Miyata, Takeshi; Harakuni, Tetsuya; Tsuboi, Takafumi; Sattabongkot, Jetsumon; Ikehara, Ayumu; Tachibana, Mayumi; Torii, Motomi; Matsuzaki, Goro; Arakawa, Takeshi

2011-01-01

The creation of subunit vaccines to prevent malaria infection has been hampered by the intrinsically weak immunogenicity of the recombinant antigens. We have developed a novel strategy to increase immune responses by creating genetic fusion proteins to target specific antigen-presenting cells (APCs). The fusion complex was composed of three physically linked molecular entities: (i) a vaccine antigen, (ii) a multimeric α-helical coiled-coil core, and (iii) an APC-targeting ligand linked to the core via a flexible linker. The vaccine efficacy of the tricomponent complex was evaluated using an ookinete surface protein of Plasmodium vivax, Pvs25, and merozoite surface protein-1 of Plasmodium yoelii. Immunization of mice with the tricomponent complex induced a robust antibody response and conferred substantial levels of P. vivax transmission blockade as evaluated by a membrane feed assay, as well as protection from lethal P. yoelii infection. The observed effect was strongly dependent on the presence of all three components physically integrated as a fusion complex. This system, designated the tricomponent immunopotentiating system (TIPS), onto which any recombinant protein antigens or nonproteinaceous substances could be loaded, may be a promising strategy for devising subunit vaccines or adjuvants against various infectious diseases, including malaria. PMID:21807905

RING1 is associated with the polycomb group protein complex and acts as a transcriptional repressor.

PubMed

Satijn, D P; Gunster, M J; van der Vlag, J; Hamer, K M; Schul, W; Alkema, M J; Saurin, A J; Freemont, P S; van Driel, R; Otte, A P

1997-07-01

The Polycomb (Pc) protein is a component of a multimeric, chromatin-associated Polycomb group (PcG) protein complex, which is involved in stable repression of gene activity. The identities of components of the PcG protein complex are largely unknown. In a two-hybrid screen with a vertebrate Pc homolog as a target, we identify the human RING1 protein as interacting with Pc. RING1 is a protein that contains the RING finger motif, a specific zinc-binding domain, which is found in many regulatory proteins. So far, the function of the RING1 protein has remained enigmatic. Here, we show that RING1 coimmunoprecipitates with a human Pc homolog, the vertebrate PcG protein BMI1, and HPH1, a human homolog of the PcG protein Polyhomeotic (Ph). Also, RING1 colocalizes with these vertebrate PcG proteins in nuclear domains of SW480 human colorectal adenocarcinoma and Saos-2 human osteosarcoma cells. Finally, we show that RING1, like Pc, is able to repress gene activity when targeted to a reporter gene. Our findings indicate that RING1 is associated with the human PcG protein complex and that RING1, like PcG proteins, can act as a transcriptional repressor.

Electron cryo-tomographic structure of cystovirus phi 12.

PubMed

Hu, Guo-Bin; Wei, Hui; Rice, William J; Stokes, David L; Gottlieb, Paul

2008-03-01

Bacteriophage phi 12 is a member of the Cystoviridae virus family and contains a genome consisting of three segments of double-stranded RNA (dsRNA). This virus family contains eight identified members, of which four have been classified in regard to their complete genomic sequence and encoded viral proteins. A phospholipid envelope that contains the integral proteins P6, P9, P10, and P13 surrounds the viral particles. In species phi 6, host infection requires binding of a multimeric P3 complex to type IV pili. In species varphi8, phi 12, and phi 13, the attachment apparatus is a heteromeric protein assembly that utilizes the rough lipopolysaccharide (rlps) as a receptor. In phi 8 the protein components are designated P3a and P3b while in species phi 12 proteins P3a and P3c have been identified in the complex. The phospholipid envelope of the cystoviruses surrounds a nucleocapsid (NC) composed of two shells. The outer shell is composed of protein P8 with a T=13 icosahedral lattice while the primary component of the inner shell is a dodecahedral frame composed of dimeric protein P1. For the current study, the 3D architecture of the intact phi 12 virus was obtained by electron cryo-tomography. The nucleocapsid appears to be centered within the membrane envelope and possibly attached to it by bridging structures. Two types of densities were observed protruding from the membrane envelope. The densities of the first type were elongated, running parallel, and closely associated to the envelope outer surface. In contrast, the second density was positioned about 12 nm above the envelope connected to it by a flexible low-density stem. This second structure formed a torroidal structure termed "the donut" and appears to inhibit BHT-induced viral envelope fusion.

Structure of a designed protein cage that self-assembles into a highly porous cube

DOE PAGES

Lai, Yen-Ting; Reading, Eamonn; Hura, Greg L.; ...

2014-11-10

Natural proteins can be versatile building blocks for multimeric, self-assembling structures. Yet, creating protein-based assemblies with specific geometries and chemical properties remains challenging. Highly porous materials represent particularly interesting targets for designed assembly. Here we utilize a strategy of fusing two natural protein oligomers using a continuous alpha-helical linker to design a novel protein that self assembles into a 750 kDa, 225 Å diameter, cube-shaped cage with large openings into a 130 Å diameter inner cavity. A crystal structure of the cage showed atomic level agreement with the designed model, while electron microscopy, native mass spectrometry, and small angle x-raymore » scattering revealed alternate assembly forms in solution. These studies show that accurate design of large porous assemblies with specific shapes is feasible, while further specificity improvements will likely require limiting flexibility to select against alternative forms. Finally, these results provide a foundation for the design of advanced materials with applications in bionanotechnology, nanomedicine and material sciences.« less

Dioleoyl-phosphatidic acid selectively binds to α-synuclein and strongly induces its aggregation.

PubMed

Mizuno, Satoru; Sasai, Hirotaka; Kume, Aiko; Takahashi, Daisuke; Satoh, Mamoru; Kado, Sayaka; Sakane, Fumio

2017-03-01

α-Synuclein (α-syn), which causally links to Parkinson's disease, binds to vesicles containing phosphatidic acid (PA). However, the effects of the fatty acyl chains of PA on its ability to bind to α-syn protein remain unclear. Intriguingly, we reveal that among several PA species, 18:1/18:1-PA is the most strongly bound PA to the α-syn protein. Moreover, 18:1/18:1-PA more strongly enhances secondary structural changes from the random coil form to the α-helical form than 16:0/18:1-PA. Furthermore, 18:1/18:1-PA more markedly accelerates generation of multimeric and proteinase K-resistant α-syn protein compared to 16:0/18:1-PA. These results indicate that among phospholipids examined so far, 18:1/18:1-PA demonstrates the strongest binding to α-syn, as well as the most effective enhancement of its secondary structural changes and aggregation formation. © 2017 Federation of European Biochemical Societies.

Increase in Mechanical Resistance to Force in a Shear-Activated Protein

NASA Astrophysics Data System (ADS)

Botello, Eric; Harris, Nolan; Choi, Huiwan; Zhou, Zhou; Bergeron, Angela; Dong, Jing-Fei; Kiang, Ching-Hwa

2009-03-01

von Willebrand factor (VWF) is the largest multimeric adhesion ligand found in human blood. Plasma VWF (pVWF) must be exposed to shear stress, like at sites of vascular injury, to be activated to bind platelets to induce blood clotting. In addition, adhesion activity of VWF is related to its polymer size, with the ultra-large form of VWF (ULVWF) being hyper-active, and forming fibers even without exposure to shear stress. We used the AFM to stretch pVWF, sheared VWF (sVWF) and ULVWF, and monitor the forces as a function of molecular extension. We showed a similar increase in force resistance to unfolding for sVWF and ULVWF when compared to pVWF. The increase in force is reduced when other molecules that are known to disrupt their fibril formation are present. Our results provide evidence that the common higher order structure of sVWF and ULVWF may affect the domain structure that causes difference in their adhesion activity compared to pVWF.

Biochemical and structural studies of the oligomerization domain of the Nipah virus phosphoprotein: evidence for an elongated coiled-coil homotrimer.

PubMed

Blocquel, David; Beltrandi, Matilde; Erales, Jenny; Barbier, Pascale; Longhi, Sonia

2013-11-01

Nipah virus (NiV) is a recently emerged severe human pathogen that belongs to the Henipavirus genus within the Paramyxoviridae family. The NiV genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that is the substrate used by the polymerase for transcription and replication. The polymerase is recruited onto the nucleocapsid via its cofactor, the phosphoprotein (P). The NiV P protein has a modular organization, with alternating disordered and ordered domains. Among these latter, is the P multimerization domain (PMD) that was predicted to adopt a coiled-coil conformation. Using both biochemical and biophysical approaches, we show that NiV PMD forms a highly stable and elongated coiled-coil trimer, a finding in striking contrast with respect to the PMDs of Paramyxoviridae members investigated so far that were all found to tetramerize. The present results therefore represent the first report of a paramyxoviral P protein forming trimers. © 2013 Elsevier Inc. All rights reserved.

Characterization of the immunoglobulin repertoire of the spiny dogfish (Squalus acanthias).

PubMed

Smith, Lauren E; Crouch, Kathryn; Cao, Wei; Müller, Mischa R; Wu, Leeying; Steven, John; Lee, Michael; Liang, Musen; Flajnik, Martin F; Shih, Heather H; Barelle, Caroline J; Paulsen, Janet; Gill, Davinder S; Dooley, Helen

2012-04-01

The cartilaginous fish (chimeras, sharks, skates and rays) are the oldest group relative to mammals in which an adaptive immune system founded upon immunoglobulins has been found. In this manuscript we characterize the immunoglobulins of the spiny dogfish (Squalus acanthias) at both the molecular and expressed protein levels. Despite the presence of hundreds of IgM clusters in this species the serum levels of this isotype are comparatively low. However, analysis of cDNA sequences and serum protein suggests microheterogeneity in the IgM heavy chains and supports the proposal that different clusters are preferentially used in the two forms (monomer or pentamer) of this isotype. We also found that the IgNAR isotype in this species exists in a previously unknown multimeric format in serum. Finally, we identified a new form of the IgW isotype (the shark IgD orthologue), in which the leader is spliced directly to the first constant domain, resulting in a molecule lacking an antigen-binding domain. Copyright © 2011 Elsevier Ltd. All rights reserved.

Purification and properties of beta-galactosidase from Aspergillus nidulans.

PubMed

Díaz, M; Pedregosa, A M; de Lucas, J R; Torralba, S; Monistrol, I F; Laborda, F

1996-12-01

Beta-Galactosidase from mycelial extract of Aspergillus nidulans has been purified by substrate affinity chromatography and used to obtain anti-beta-galactosidase polyclonal antibodies. A. nidulans growing in lactose as carbon source synthesizes one active form of beta-galactosidase which seems to be a multimeric enzyme of 450 kDa composed of monomers with 120 and 97 kDa. Although the enzyme was not released to the culture medium, some enzymatic activity was detected in a cell-wall extract, thus suggesting that it can be an extracellular enzyme. Beta-Galactosidase of A. nidulans is a very unstable enzyme with an optimum pH value of 7.5 and an optimum temperature of 30 degrees C. It was only active against beta-galactoside substrates like lactose and p-nitrophenyl-beta-D-galactoside (PNPG).

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

PubMed Central

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

2012-01-01

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

Group additivity-Pourbaix diagrams advocate thermodynamically stable nanoscale clusters in aqueous environments

PubMed Central

Wills, Lindsay A.; Qu, Xiaohui; Chang, I-Ya; Mustard, Thomas J. L.; Keszler, Douglas A.; Persson, Kristin A.; Cheong, Paul Ha-Yeon

2017-01-01

The characterization of water-based corrosion, geochemical, environmental and catalytic processes rely on the accurate depiction of stable phases in a water environment. The process is aided by Pourbaix diagrams, which map the equilibrium solid and solution phases under varying conditions of pH and electrochemical potential. Recently, metastable or possibly stable nanometric aqueous clusters have been proposed as intermediate species in non-classical nucleation processes. Herein, we describe a Group Additivity approach to obtain Pourbaix diagrams with full consideration of multimeric cluster speciation from computations. Comparisons with existing titration results from experiments yield excellent agreement. Applying this Group Additivity-Pourbaix approach to Group 13 elements, we arrive at a quantitative evaluation of cluster stability, as a function of pH and concentration, and present compelling support for not only metastable but also thermodynamically stable multimeric clusters in aqueous solutions. PMID:28643782

The ability of multimerized cyclophilin A to restrict retrovirus infection

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

Javanbakht, Hassan; Diaz-Griffero, Felipe; Yuan Wen

2007-10-10

In owl monkeys, the typical retroviral restriction factor of primates, TRIM5{alpha}, is replaced by TRIMCyp. TRIMCyp consists of the TRIM5 RING, B-box 2 and coiled-coil domains, as well as the intervening linker regions, fused with cyclophilin A. TRIMCyp restricts infection of retroviruses, such as human immunodeficiency virus (HIV-1) and feline immunodeficiency virus (FIV), with capsids that can bind cyclophilin A. The TRIM5 coiled coil promotes the trimerization of TRIMCyp. Here we show that cyclophilin A that is oligomeric as a result of fusion with a heterologous multimer exhibits substantial antiretroviral activity. The addition of the TRIM5 RING, B-box 2 andmore » Linker 2 to oligomeric cyclophilin A generated a protein with antiretroviral activity approaching that of wild-type TRIMCyp. Multimerization increased the binding of cyclophilin A to the HIV-1 capsid, promoting accelerated uncoating of the capsid and restriction of infection.« less

Group additivity-Pourbaix diagrams advocate thermodynamically stable nanoscale clusters in aqueous environments

NASA Astrophysics Data System (ADS)

Wills, Lindsay A.; Qu, Xiaohui; Chang, I.-Ya; Mustard, Thomas J. L.; Keszler, Douglas A.; Persson, Kristin A.; Cheong, Paul Ha-Yeon

2017-06-01

The characterization of water-based corrosion, geochemical, environmental and catalytic processes rely on the accurate depiction of stable phases in a water environment. The process is aided by Pourbaix diagrams, which map the equilibrium solid and solution phases under varying conditions of pH and electrochemical potential. Recently, metastable or possibly stable nanometric aqueous clusters have been proposed as intermediate species in non-classical nucleation processes. Herein, we describe a Group Additivity approach to obtain Pourbaix diagrams with full consideration of multimeric cluster speciation from computations. Comparisons with existing titration results from experiments yield excellent agreement. Applying this Group Additivity-Pourbaix approach to Group 13 elements, we arrive at a quantitative evaluation of cluster stability, as a function of pH and concentration, and present compelling support for not only metastable but also thermodynamically stable multimeric clusters in aqueous solutions.

The GP(Y/F) Domain of TF1 Integrase Multimerizes when Present in a Fragment, and Substitutions in This Domain Reduce Enzymatic Activity of the Full-length Protein*S⃞

PubMed Central

Ebina, Hirotaka; Chatterjee, Atreyi Ghatak; Judson, Robert L.; Levin, Henry L.

2008-01-01

Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the γ-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining. PMID:18397885

The GP(Y/F) domain of TF1 integrase multimerizes when present in a fragment, and substitutions in this domain reduce enzymatic activity of the full-length protein.

PubMed

Ebina, Hirotaka; Chatterjee, Atreyi Ghatak; Judson, Robert L; Levin, Henry L

2008-06-06

Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the gamma-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining.

Structural dynamics of native and V260E mutant C-terminal domain of HIV-1 integrase

NASA Astrophysics Data System (ADS)

Sangeetha, Balasubramanian; Muthukumaran, Rajagopalan; Amutha, Ramaswamy

2015-04-01

The C-terminal domain (CTD) of HIV-1 integrase is a five stranded β-barrel resembling an SH3 fold. Mutational studies on isolated CTD and full-length IN have reported V260E mutant as either homo-dimerization defective or affecting the stability and folding of CTD. In this study, molecular dynamics simulation techniques were used to unveil the effect of V260E mutation on isolated CTD monomer and dimer. Both monomeric and dimeric forms of wild type and V260E mutant are highly stable during the simulated period. However, the stabilizing π-stacking interaction between Trp243 and Trp243' at the dimer interface is highly disturbed in CTD-V260E (>6 Å apart). The loss in entropy for dimerization is -30 and -25 kcal/mol for CTD-wt and CTD-V260E respectively signifying a weak hydrophobic interaction and its perturbation in CTD-V260E. The mutant Glu260 exhibits strong attraction/repulsion with all the basic/acidic residues of CTD. In addition to this, the dynamics of CTD-wild type and V260E monomers at 498 K was analyzed to elucidate the effect of V260E mutation on CTD folding. Increase in SASA and reduction in the number of contacts in CTD-V260E during simulation highlights the instability caused by the mutation. In general, V260E mutation affects both multimerization and protein folding with a pronounced effect on protein folding rather than multimerization. This study emphasizes the importance of the hydrophobic nature and SH3 fold of CTD in proper functioning of HIV integrase and perturbing this nature would be a rational approach toward designing more selective and potent allosteric anti-HIV inhibitors.

miR-ID: A novel, circularization-based platform for detection of microRNAs

PubMed Central

Kumar, Pavan; Johnston, Brian H.; Kazakov, Sergei A.

2011-01-01

MicroRNAs (miRNAs) are important regulators of gene expression and have great potential as biomarkers, prognostic indicators, and therapeutic targets. Determining the expression patterns of these molecules is essential for elucidating their biogenesis, regulation, relation to disease, and response to therapy. Although PCR-based assays are commonly used for expression profiling of miRNAs, the small size, sequence heterogeneity, and (in some cases) end modifications of miRNAs constrain the performance of existing PCR methods. Here we introduce miR-ID, a novel method that avoids these constraints while providing superior sensitivity and sequence specificity at a lower cost. It also has the unique ability to differentiate unmodified small RNAs from those carrying 2′-OMe groups at their 3′-ends while detecting both forms. miR-ID is comprised of the following steps: (1) circularization of the miRNA by a ligase; (2) reverse transcription of the circularized miRNA (RTC), producing tandem repeats of a DNA sequence complementary to the miRNA; and (3) qPCR amplification of segments of this multimeric cDNA using 5′-overlapping primers and a nonspecific dye such as SYBR Green. No chemically modified probes (e.g., TaqMan) or primers (e.g., LNA) are required. The circular RNA and multimeric cDNA templates provide unmatched flexibility in the positioning of primers, which may include straddling the boundaries between these repetitive miRNA sequences. miR-ID is based on new findings that are themselves of general interest, including reverse transcription of small RNA circles and the use of 5′-overlapping primers for detection of repetitive sequences by qPCR. PMID:21169480

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

PubMed Central

Arturo, Emilia C.; Gupta, Kushol; Héroux, Annie; Stith, Linda; Cross, Penelope J.; Parker, Emily J.; Loll, Patrick J.; Jaffe, Eileen K.

2016-01-01

Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73–82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype–phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype–phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate. PMID:26884182

Overexpression of the VSSC-associated CAM, β-2, enhances LNCaP cell metastasis associated behavior.

PubMed

Jansson, Keith H; Lynch, Jill E; Lepori-Bui, Nadia; Czymmek, Kirk J; Duncan, Randall L; Sikes, Robert A

2012-07-01

Prostate cancer (PCa) is the second-leading cause of cancer death in American men. This is due largely to the "silent" nature of the disease until it has progressed to a highly metastatic and castrate resistant state. Voltage sensitive sodium channels (VSSCs) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two β subunits. The β-subunits modulate surface expression and gating kinetics of the channels but also have inherent cell adhesion molecule (CAM) functions. We hypothesize that PCa cells use VSSC β-subunits as CAMs during PCa progression and metastasis. We overexpressed the beta-2 isoform as a C-terminal fusion protein with enhanced cyan fluorescence protein (ECFP) in the weakly metastatic LNCaP cells. The effect of beta-2 overexpression on cell morphology was examined using confocal microscopy while metastasis-associated behavior was tested by performing several in vitro metastatic functional assays and in vivo subcutaneous tumor studies. We found that cells overexpressing beta-2 (2BECFP) converted to a bipolar fibroblastic morphology. 2BECFP cells were more adhesive than control (ECFP) to vitronectin (twofold) and Matrigel® (1.3-fold), more invasive through Matrigel® (3.6-fold in 72 hr), and had enhanced migration (2.1-fold in 96 hr) independent of proliferation in wound-healing assays. In contrast, 2BECFP cells have a reduced tumor-take and tumor volume in vivo even though the overexpression of beta-2 was maintained. Functional overexpression of VSSC β-subunits in PCa may be one mechanism leading to increased metastatic behavior while decreasing the ability to form localized tumor masses. Copyright © 2011 Wiley Periodicals, Inc.

Laboratory diagnosis of von Willebrand disease type 1/2E (2A subtype IIE), type 1 Vicenza and mild type 1 caused by mutations in the D3, D4, B1-B3 and C1-C2 domains of the von Willebrand factor gene. Role of von Willebrand factor multimers and the von Willebrand factor propeptide/antigen ratio.

PubMed

Gadisseur, Alain; Berneman, Zwi; Schroyens, Wilfried; Michiels, Jan Jacques

2009-01-01

Autosomal dominant von Willebrand disease (VWD) type 1/2E is a quantitative/qualitative defect in the von Willebrand factor (VWF) caused by heterozygous cysteine and non-cysteine mutations in the D3 domain of the VWF gene and results in a secretion-multimerization-clearance defect in mutant VWF with the loss of large VWF multimers not due to proteolysis. The multimers of patients with dominant VWD type 1/2E due to mutations in the D3 domain show an aberrant triplet structure with lack of outer bands but with pronounced inner bands of the triplet structure combined with a relative decrease in large multimers reflecting heterozygosity for multimerization defects. There is a good response to desmopressin (DDAVP) followed by rapid clearance of VWF:antigen (Ag), factor VIII coagulant activity (FVIII:C) and VWF:ristocetin cofactor activity (RCo) as the main cause of VWD type 1 or 2 with typical 2E multimeric pattern (VWD type 1/2E). Cysteine mutations in the D3 domains (C1130, C1149 and C1190) show pronounced features of VWD 1/2E with the relative loss of large and relative increase in small VWF multimers with abnormal triplet structure in heterozygotes. Such abnormalities are less pronounced in patients with a milder form of VWD type 1 due to non-cysteine mutations W1144G, T1156M and W1120S in the D3 domain. VWD type 1 Vicenza is caused by the R1205H mutation in the D3 domain and characterized by equally low levels of FVIII:C, VWF:Ag and VWF:RCo. The response to DDAVP in VWD Vicenza is good for FVIII:C, VWF:Ag and VWF:RCo, which is followed by a rapid clearance in less than a few hours of FVIII:C and VWF parameters. The ratios for FVIII:C/VWF:Ag, VWF:RCo/Ag and VWF:CB/Ag remain normal before and after DDAVP indicating that VWD Vicenza clearly differs from VWD type 1, 1/2E and 2M. A new set of missense mutations in D4, B1-B3 and C1-C2 domains has been discovered as the cause of a mild VWD type 1 secretion defect with normal VWF multimers or smeary VWF multimeric pattern. Cysteine mutations in exons 38, 40, 42 and 43 (D4, B1-B3 and C1 domain), show smeary patterns (either smf or sm), with the presence of large VWF multimers and a laboratory phenotype of mild VWD type 1 with variable penetrance of bleeding manifestations. Recent studies showed that the ratio of VWF propeptide (pp) to VWF:Ag can be used to predict a shorter than normal half-life for VWF:Ag. There is a strong inverse correlation between rapid clearance of VWF:Ag after DDAVP and increased VWFpp/Ag ratios >10 in VWD type 1 Vicenza, and >2 in VWD type 1/2E but normal or slightly increased (1-<2) VWFpp/Ag ratios in mild-type VWD due to nonsense or missense mutations in the D1, D2, D4, B and C domains. Copyright (c) 2009 S. Karger AG, Basel.

Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer.

PubMed

Bar-Peled, Liron; Kemper, Esther K; Suciu, Radu M; Vinogradova, Ekaterina V; Backus, Keriann M; Horning, Benjamin D; Paul, Thomas A; Ichu, Taka-Aki; Svensson, Robert U; Olucha, Jose; Chang, Max W; Kok, Bernard P; Zhu, Zhou; Ihle, Nathan T; Dix, Melissa M; Jiang, Ping; Hayward, Matthew M; Saez, Enrique; Shaw, Reuben J; Cravatt, Benjamin F

2017-10-19

The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers. Copyright © 2017 Elsevier Inc. All rights reserved.

Crystal structure of B acillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity: AtxA multimerization, phosphorylation and activity

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

Hammerstrom, Troy G.; Horton, Lori B.; Swick, Michelle C.

2014-12-30

The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (HisAsp) and phosphoablative (HisAla) amino acid changes for activitymore » in B.anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.« less

Distinct Contributions of T1R2 and T1R3 Taste Receptor Subunits to the Detection of Sweet Stimuli

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

Nie,Y.; Vigues, S.; Hobbs, J.

2005-01-01

The molecular mechanisms by which G protein-coupled receptor (GPCR)-type chemosensory receptors of animals selectively interact with their cognate ligands remain poorly understood. There is growing evidence that many chemosensory receptors exist in multimeric complexes, though little is known about the relative contributions of individual subunits to receptor functions. This study showed that each of the two subunits in the mammalian heteromeric T1R2:T1R3 sweet taste receptor binds sweet stimuli, though with distinct affinities and conformational changes. Furthermore, ligand affinities for T1R3 are drastically reduced by the introduction of a single amino acid change associated with decreased sweet taste sensitivity in mice.more » Thus, individual T1R subunits increase the receptive range of the sweet taste receptor, offering a functional mechanism for phenotypic variations in sweet taste.« less

Allosteric mechanism of water channel gating by Ca2+–calmodulin

PubMed Central

Reichow, Steve L.; Clemens, Daniel M.; Freites, J. Alfredo; Németh-Cahalan, Karin L.; Heyden, Matthias; Tobias, Douglas J.; Hall, James E.; Gonen, Tamir

2013-01-01

Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, our understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudo-atomic structure of full-length mammalian aquaporin-0 (AQP0, Bos Taurus) in complex with CaM using electron microscopy to understand how this signaling protein modulates water channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits. PMID:23893133

Structure and signalling functions of C3 receptors on human B cells.

PubMed

Frade, R

1990-03-01

CR1 (C3b receptor) and CR2 (C3d/EBV receptor) are two C3 receptors expressed on B lymphocytes. CR1 and CR2 have structural similarities and their cross-linking at the B cell surface by antibodies or specific ligands in multimeric forms induce B cell activation. However, activation of human B cells through cell surface interactions or by intracellular protein kinase C activators leads to phosphorylation of CR2 but not CR1. CR2 is phosphorylated on serine and tyrosine residues. Analysis of post-membrane events associated with CR2 revealed intracellular interactions of CR2 with p53, a plasma membrane anti-oncogene-encoded phosphoprotein, and with p120, a nuclear phosphoribonucleoprotein. These intracellular interactions probably represent important steps in the signalling functions of CR2.

Mechanosensitive channels in bacteria as membrane tension reporters

NASA Technical Reports Server (NTRS)

Sukharev, S.

1999-01-01

The purpose of this short review is to discuss recent data on the molecular structure and mechanism of gating of MscL, a mechanosensitive channel of large conductance from Escherichia coli. MscL is the first isolated molecule shown to convert mechanical stress of the membrane into a simple response, the opening of a large aqueous pore. The functional complex appears to be a stable homo-pentamer of 15-kDa subunits, the gating transitions in which are driven by stretch forces conveyed through the lipid bilayer. We have measured the open probability of MscL and the kinetics of transitions as a function of membrane tension. The parameters extracted from the single-channel current recordings and dose-response curves such as the energy difference between the closed, open, and intermediate conducting states, and the transition-related changes in protein dimensions suggest a large conformational rearrangement of the channel complex. The estimations show that in native conditions MscL openings could be driven primarily by forces of osmotic nature. The thermodynamic and spatial parameters reasonably correlate with the available data on the structure of a single MscL subunit and multimeric organization of the complex. Combined with the functional analysis of mutations, these data give grounds to hypotheses on the nature of the channel mechanosensitivity.

A genetic screen for terminator function in yeast identifies a role for a new functional domain in termination factor Nab3

PubMed Central

Loya, Travis J.; O’Rourke, Thomas W.; Reines, Daniel

2012-01-01

The yeast IMD2 gene encodes an enzyme involved in GTP synthesis. Its expression is controlled by guanine nucleotides through a set of alternate start sites and an intervening transcriptional terminator. In the off state, transcription results in a short non-coding RNA that starts upstream of the gene. Transcription terminates via the Nrd1-Nab3-Sen1 complex and is degraded by the nuclear exosome. Using a sensitive terminator read-through assay, we identified trans-acting Terminator Override (TOV) genes that operate this terminator. Four genes were identified: the RNA polymerase II phosphatase SSU72, the RNA polymerase II binding protein PCF11, the TRAMP subunit TRF4 and the hnRNP-like, NAB3. The TOV phenotype can be explained by the loss of function of these gene products as described in models in which termination and RNA degradation are coupled to the phosphorylation state of RNA polymerase II's repeat domain. The most interesting mutations were those found in NAB3, which led to the finding that the removal of merely three carboxy-terminal amino acids compromised Nab3's function. This region of previously unknown function is distant from the protein's well-known RNA binding and Nrd1 binding domains. Structural homology modeling suggests this Nab3 ‘tail’ forms an α-helical multimerization domain that helps assemble it onto an RNA substrate. PMID:22564898

Tertiary model of a plant cellulose synthase

PubMed Central

Sethaphong, Latsavongsakda; Haigler, Candace H.; Kubicki, James D.; Zimmer, Jochen; Bonetta, Dario; DeBolt, Seth; Yingling, Yaroslava G.

2013-01-01

A 3D atomistic model of a plant cellulose synthase (CESA) has remained elusive despite over forty years of experimental effort. Here, we report a computationally predicted 3D structure of 506 amino acids of cotton CESA within the cytosolic region. Comparison of the predicted plant CESA structure with the solved structure of a bacterial cellulose-synthesizing protein validates the overall fold of the modeled glycosyltransferase (GT) domain. The coaligned plant and bacterial GT domains share a six-stranded β-sheet, five α-helices, and conserved motifs similar to those required for catalysis in other GT-2 glycosyltransferases. Extending beyond the cross-kingdom similarities related to cellulose polymerization, the predicted structure of cotton CESA reveals that plant-specific modules (plant-conserved region and class-specific region) fold into distinct subdomains on the periphery of the catalytic region. Computational results support the importance of the plant-conserved region and/or class-specific region in CESA oligomerization to form the multimeric cellulose–synthesis complexes that are characteristic of plants. Relatively high sequence conservation between plant CESAs allowed mapping of known mutations and two previously undescribed mutations that perturb cellulose synthesis in Arabidopsis thaliana to their analogous positions in the modeled structure. Most of these mutation sites are near the predicted catalytic region, and the confluence of other mutation sites supports the existence of previously undefined functional nodes within the catalytic core of CESA. Overall, the predicted tertiary structure provides a platform for the biochemical engineering of plant CESAs. PMID:23592721

Differential Effects of Leptin and Adiponectin in Endothelial Angiogenesis

PubMed Central

Adya, Raghu; Tan, Bee K.; Randeva, Harpal S.

2015-01-01

Obesity is a major health burden with an increased risk of cardiovascular morbidity and mortality. Endothelial dysfunction is pivotal to the development of cardiovascular disease (CVD). In relation to this, adipose tissue secreted factors termed “adipokines” have been reported to modulate endothelial dysfunction. In this review, we focus on two of the most abundant circulating adipokines, that is, leptin and adiponectin, in the development of endothelial dysfunction. Leptin has been documented to influence a multitude of organ systems, that is, central nervous system (appetite regulation, satiety factor) and cardiovascular system (endothelial dysfunction leading to atherosclerosis). Adiponectin, circulating at a much higher concentration, exists in different molecular weight forms, essentially made up of the collagenous fraction and a globular domain, the latter being investigated minimally for its involvement in proinflammatory processes including activation of NF-κβ and endothelial adhesion molecules. The opposing actions of the two forms of adiponectin in endothelial cells have been recently demonstrated. Additionally, a local and systemic change to multimeric forms of adiponectin has gained importance. Thus detailed investigations on the potential interplay between these adipokines would likely result in better understanding of the missing links connecting CVD, adipokines, and obesity. PMID:25650072

Single-Molecule Manipulation Studies of a Mechanically Activated Protein

NASA Astrophysics Data System (ADS)

Botello, Eric; Harris, Nolan; Choi, Huiwan; Bergeron, Angela; Dong, Jing-Fei; Kiang, Ching-Hwa

2009-10-01

Plasma von Willebrand factor (pVWF) is the largest multimeric adhesion ligand found in human blood and must be adhesively activated by exposure to shear stress, like at sites of vascular injury, to initiate blood clotting. Sheared pVWF (sVWF) will undergo a conformational change from a loose tangled coil to elongated strings forming adhesive fibers by binding with other sVWF. VWF's adhesion activity is also related to its length, with the ultra-large form of VWF (ULVWF) being hyper-actively adhesive without exposure to shear stress; it has also been shown to spontaneously form fibers. We used single molecule manipulation techniques with the AFM to stretch pVWF, sVWF and ULVWF and monitor the forces as a function of molecular extension. We showed a similar increase in resistance to unfolding for sVWF and ULVWF when compared to pVWF. This mechanical resistance to forced unfolding is reduced when other molecules known to disrupt their fibril formation are present. Our results show that sVWF and ULVWF domains unfold at higher forces than pVWF, which is consistent with the hypothesis that shear stress induces lateral association that alters adhesion activity of pVWF.

Crystallization and preliminary X-ray crystallographic study of a 3.8-MDa respiratory supermolecule hemocyanin.

PubMed

Matsuno, Asuka; Gai, Zuoqi; Tanaka, Miyuki; Kato, Koji; Kato, Sanae; Katoh, Tsuyoshi; Shimizu, Takeshi; Yoshioka, Takeya; Kishimura, Hideki; Tanaka, Yoshikazu; Yao, Min

2015-06-01

Many molluscs transport oxygen using a very large cylindrical multimeric copper-containing protein named hemocyanin. The molluscan hemocyanin forms a decamer (cephalopods) or multidecamer (gastropods) of approximately 330-450kDa subunits, resulting in a molecular mass >3.3MDa. Therefore, molluscan hemocyanin is one of the largest proteins. The reason why these organisms use such a large supermolecule for oxygen transport remains unclear. Atomic-resolution X-ray crystallographic analysis is necessary to unveil the detailed molecular structure of this mysterious large molecule. However, its propensity to dissociate in solution has hampered the crystallization of its intact form. In the present study, we successfully obtained the first crystals of an intact decameric molluscan hemocyanin. The diffraction dataset at 3.0-Å resolution was collected by merging the datasets of two isomorphic crystals. Electron microscopy analysis of the dissolved crystals revealed cylindrical particles. Furthermore, self-rotation function analysis clearly showed the presence of a fivefold symmetry with several twofold symmetries perpendicular to the fivefold axis. The absorption spectrum of the crystals showed an absorption peak around 345nm. These results indicated that the crystals contain intact hemocyanin decamers in the oxygen-bound form. Copyright © 2015 Elsevier Inc. All rights reserved.

Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation.

PubMed

Jurado, Kellie A; Wang, Hao; Slaughter, Alison; Feng, Lei; Kessl, Jacques J; Koh, Yasuhiro; Wang, Weifeng; Ballandras-Colas, Allison; Patel, Pratiq A; Fuchs, James R; Kvaratskhelia, Mamuka; Engelman, Alan

2013-05-21

Integration is essential for HIV-1 replication, and the viral integrase (IN) protein is an important therapeutic target. Allosteric IN inhibitors (ALLINIs) that engage the IN dimer interface at the binding site for the host protein lens epithelium-derived growth factor (LEDGF)/transcriptional coactivator p75 are an emerging class of small molecule antagonists. Consistent with the inhibition of a multivalent drug target, ALLINIs display steep antiviral dose-response curves ex vivo. ALLINIs multimerize IN protein and concordantly block its assembly with viral DNA in vitro, indicating that the disruption of two integration-associated functions, IN catalysis and the IN-LEDGF/p75 interaction, determines the multimode mechanism of ALLINI action. We now demonstrate that ALLINI potency is unexpectedly accounted for during the late phase of HIV-1 replication. The compounds promote virion IN multimerization and, reminiscent of class II IN mutations, block the formation of the electron-dense viral core and inhibit reverse transcription and integration in subsequently infected target cells. Mature virions are recalcitrant to ALLINI treatment, and compound potency during virus production is independent of the level of LEDGF/p75 expression. We conclude that cooperative multimerization of IN by ALLINIs together with the inability for LEDGF/p75 to effectively engage the virus during its egress from cells underscores the multimodal mechanism of ALLINI action. Our results highlight the versatile nature of allosteric inhibitors to primarily inhibit viral replication at a step that is distinct from the catalytic requirement for the target enzyme. The vulnerability of IN to small molecules during the late phase of HIV-1 replication unveils a pharmacological Achilles' heel for exploitation in clinical ALLINI development.

Dimerization of the voltage-sensing phosphatase controls its voltage-sensing and catalytic activity.

PubMed

Rayaprolu, Vamseedhar; Royal, Perrine; Stengel, Karen; Sandoz, Guillaume; Kohout, Susy C

2018-05-07

Multimerization is a key characteristic of most voltage-sensing proteins. The main exception was thought to be the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP). In this study, we show that multimerization is also critical for Ci-VSP function. Using coimmunoprecipitation and single-molecule pull-down, we find that Ci-VSP stoichiometry is flexible. It exists as both monomers and dimers, with dimers favored at higher concentrations. We show strong dimerization via the voltage-sensing domain (VSD) and weak dimerization via the phosphatase domain. Using voltage-clamp fluorometry, we also find that VSDs cooperate to lower the voltage dependence of activation, thus favoring the activation of Ci-VSP. Finally, using activity assays, we find that dimerization alters Ci-VSP substrate specificity such that only dimeric Ci-VSP is able to dephosphorylate the 3-phosphate from PI(3,4,5)P 3 or PI(3,4)P 2 Our results indicate that dimerization plays a significant role in Ci-VSP function. © 2018 Rayaprolu et al.

Salt Bridge Rearrangement (SaBRe) Explains the Dissociation Behavior of Noncovalent Complexes

NASA Astrophysics Data System (ADS)

Loo, Rachel R. Ogorzalek; Loo, Joseph A.

2016-06-01

Native electrospray ionization-mass spectrometry, with gas-phase activation and solution compositions that partially release subcomplexes, can elucidate topologies of macromolecular assemblies. That so much complexity can be preserved in gas-phase assemblies is remarkable, although a long-standing conundrum has been the differences between their gas- and solution-phase decompositions. Collision-induced dissociation of multimeric noncovalent complexes typically distributes products asymmetrically (i.e., by ejecting a single subunit bearing a large percentage of the excess charge). That unexpected behavior has been rationalized as one subunit "unfolding" to depart with more charge. We present an alternative explanation based on heterolytic ion-pair scission and rearrangement, a mechanism that inherently partitions charge asymmetrically. Excessive barriers to dissociation are circumvented in this manner, when local charge rearrangements access a lower-barrier surface. An implication of this ion pair consideration is that stability differences between high- and low-charge state ions usually attributed to Coulomb repulsion may, alternatively, be conveyed by attractive forces from ion pairs (salt bridges) stabilizing low-charge state ions. Should the number of ion pairs be roughly inversely related to charge, symmetric dissociations would be favored from highly charged complexes, as observed. Correlations between a gas-phase protein's size and charge reflect the quantity of restraining ion pairs. Collisionally-facilitated salt bridge rearrangement (SaBRe) may explain unusual size "contractions" seen for some activated, low charge state complexes. That some low-charged multimers preferentially cleave covalent bonds or shed small ions to disrupting noncovalent associations is also explained by greater ion pairing in low charge state complexes.

Salt Bridge Rearrangement (SaBRe) Explains the Dissociation Behavior of Noncovalent Complexes.

PubMed

Loo, Rachel R Ogorzalek; Loo, Joseph A

2016-06-01

Native electrospray ionization-mass spectrometry, with gas-phase activation and solution compositions that partially release subcomplexes, can elucidate topologies of macromolecular assemblies. That so much complexity can be preserved in gas-phase assemblies is remarkable, although a long-standing conundrum has been the differences between their gas- and solution-phase decompositions. Collision-induced dissociation of multimeric noncovalent complexes typically distributes products asymmetrically (i.e., by ejecting a single subunit bearing a large percentage of the excess charge). That unexpected behavior has been rationalized as one subunit "unfolding" to depart with more charge. We present an alternative explanation based on heterolytic ion-pair scission and rearrangement, a mechanism that inherently partitions charge asymmetrically. Excessive barriers to dissociation are circumvented in this manner, when local charge rearrangements access a lower-barrier surface. An implication of this ion pair consideration is that stability differences between high- and low-charge state ions usually attributed to Coulomb repulsion may, alternatively, be conveyed by attractive forces from ion pairs (salt bridges) stabilizing low-charge state ions. Should the number of ion pairs be roughly inversely related to charge, symmetric dissociations would be favored from highly charged complexes, as observed. Correlations between a gas-phase protein's size and charge reflect the quantity of restraining ion pairs. Collisionally-facilitated salt bridge rearrangement (SaBRe) may explain unusual size "contractions" seen for some activated, low charge state complexes. That some low-charged multimers preferentially cleave covalent bonds or shed small ions to disrupting noncovalent associations is also explained by greater ion pairing in low charge state complexes. Graphical Abstract ᅟ.

Cellular localization and detergent dependent oligomerization of rice allene oxide synthase-1.

PubMed

Yoeun, Sereyvath; Kim, Jeong-Il; Han, Oksoo

2015-01-01

Allene oxide synthase-1 from Oryza sativa (OsAOS1) localizes to the chloroplast, but lacks a putative chloroplast targeting sequence typically found in dicot AOS. Here, kinetic parameters and the oligomerization state/subunit composition of OsAOS1 were characterized in vitro in the absence or presence of detergent micelles. The catalytic efficiency (k(cat)/K(m)) of OsAOS1 reached a maximum near the critical micelle concentration for polyoxyethylene 10 tridecyl ether. Native gel analysis showed that OsAOS1 exists as a multimer in the absence of detergent micelles. The multimeric form of OsAOS1 was stably cross-linked in the absence of detergents, while only monomeric OsAOS1 was detected in the presence of detergent micelles. Gel filtration analysis indicated that the oligomeric state of OsAOS1 depends strongly on the detergents and that the monomer becomes the predominant form in the presence of detergent micelles. These data suggest that the detergent-dependent oligomeric state of OsAOS1 is an important factor for the regulation of its catalytic efficiency.

GSK3-mediated raptor phosphorylation supports amino-acid-dependent mTORC1-directed signalling

PubMed Central

Stretton, Clare; Hoffmann, Thorsten M.; Munson, Michael J.; Prescott, Alan; Taylor, Peter M.; Ganley, Ian G.; Hundal, Harinder S.

2015-01-01

The mammalian or mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a ubiquitously expressed multimeric protein kinase complex that integrates nutrient and growth factor signals for the co-ordinated regulation of cellular metabolism and cell growth. Herein, we demonstrate that suppressing the cellular activity of glycogen synthase kinase-3 (GSK3), by use of pharmacological inhibitors or shRNA-mediated gene silencing, results in substantial reduction in amino acid (AA)-regulated mTORC1-directed signalling, as assessed by phosphorylation of multiple downstream mTORC1 targets. We show that GSK3 regulates mTORC1 activity through its ability to phosphorylate the mTOR-associated scaffold protein raptor (regulatory-associated protein of mTOR) on Ser859. We further demonstrate that either GSK3 inhibition or expression of a S859A mutated raptor leads to reduced interaction between mTOR and raptor and under these circumstances, irrespective of AA availability, there is a consequential loss in phosphorylation of mTOR substrates, such as p70S6K1 (ribosomal S6 kinase 1) and uncoordinated-51-like kinase (ULK1), which results in increased autophagic flux and reduced cellular proliferation. PMID:26348909

Modulation of cardiac contractility by the phospholamban/SERCA2a regulatome.

PubMed

Kranias, Evangelia G; Hajjar, Roger J

2012-06-08

Heart disease remains the leading cause of death and disability in the Western world. Current therapies aim at treating the symptoms rather than the subcellular mechanisms, underlying the etiology and pathological remodeling in heart failure. A universal characteristic, contributing to the decreased contractile performance in human and experimental failing hearts, is impaired calcium sequestration into the sarcoplasmic reticulum (SR). SR calcium uptake is mediated by a Ca(2+)-ATPase (SERCA2), whose activity is reversibly regulated by phospholamban (PLN). Dephosphorylated PLN is an inhibitor of SERCA and phosphorylation of PLN relieves this inhibition. However, the initial simple view of a PLN/SERCA regulatory complex has been modified by our recent identification of SUMO, S100 and the histidine-rich Ca-binding protein as regulators of SERCA activity. In addition, PLN activity is regulated by 2 phosphoproteins, the inhibitor-1 of protein phosphatase 1 and the small heat shock protein 20, which affect the overall SERCA-mediated Ca-transport. This review will highlight the regulatory mechanisms of cardiac contractility by the multimeric SERCA/PLN-ensemble and the potential for new therapeutic avenues targeting this complex by using small molecules and gene transfer methods.

TAPBPR bridges UDP-glucose:glycoprotein glucosyltransferase 1 onto MHC class I to provide quality control in the antigen presentation pathway

PubMed Central

Neerincx, Andreas; Hermann, Clemens; Antrobus, Robin; van Hateren, Andy; Cao, Huan; Trautwein, Nico; Stevanović, Stefan; Elliott, Tim; Deane, Janet E; Boyle, Louise H

2017-01-01

Recently, we revealed that TAPBPR is a peptide exchange catalyst that is important for optimal peptide selection by MHC class I molecules. Here, we asked whether any other co-factors associate with TAPBPR, which would explain its effect on peptide selection. We identify an interaction between TAPBPR and UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1), a folding sensor in the calnexin/calreticulin quality control cycle that is known to regenerate the Glc1Man9GlcNAc2 moiety on glycoproteins. Our results suggest the formation of a multimeric complex, dependent on a conserved cysteine at position 94 in TAPBPR, in which TAPBPR promotes the association of UGT1 with peptide-receptive MHC class I molecules. We reveal that the interaction between TAPBPR and UGT1 facilities the reglucosylation of the glycan on MHC class I molecules, promoting their recognition by calreticulin. Our results suggest that in addition to being a peptide editor, TAPBPR improves peptide optimisation by promoting peptide-receptive MHC class I molecules to associate with the peptide-loading complex. DOI: http://dx.doi.org/10.7554/eLife.23049.001 PMID:28425917

The Thiamin Pyrophosphate-Motif

NASA Technical Reports Server (NTRS)

Dominiak, P.; Ciszak, E.

2003-01-01

Using databases the authors have identified a common thiamin pyrophosphate (TPP)-motif in the family of functionally diverse TPP-dependent enzymes. This common motif consists of multimeric organization of subunits and two catalytic centers. Each catalytic center (PP:PYR) is formed at the interface of the PP-domain binding the magnesium ion, pyrophosphate and amhopyrimidine ring of TPP, and the PYR-domain binding the aminopyrimidine ring of that cofactor. A pair of these catalytic centers constitutes the catalytic core (PP:PYR)(sub 2) within these enzymes. Analysis of the structural elements of this catalytic core reveals novel definition of the common amino acid sequences, which are GXPhiX(sub 4)(G)PhiXXGQ and GDGX(sub 25-30)NN in the PP-domain, and the EX(sub 4)(G)PhiXXGPhi in the PYR-domain, where Phi corresponds to a hydrophobic amino acid. This TPP-motif provides a novel tool for annotation of TPP-dependent enzymes useful in advancing functional proteomics.

Potential Roles of Amiloride-Sensitive Sodium Channels in Cancer Development.

PubMed

Xu, Siguang; Liu, Cui; Ma, Yana; Ji, Hong-Long; Li, Xiumin

2016-01-01

The ENaC/degenerin ion channel superfamily includes the amiloride-sensitive epithelial sodium channel (ENaC) and acid sensitive ionic channel (ASIC). ENaC is a multimeric ion channel formed by heteromultimeric membrane glycoproteins, which participate in a multitude of biological processes by mediating the transport of sodium (Na(+)) across epithelial tissues such as the kidney, lungs, bladder, and gut. Aberrant ENaC functions contribute to several human disease states including pseudohypoaldosteronism, Liddle syndrome, cystic fibrosis, and salt-sensitive hypertension. Increasing evidence suggests that ion channels not only regulate ion homeostasis and electric signaling in excitable cells but also play important roles in cancer cell behaviors such as proliferation, apoptosis, invasion, and migration. Indeed, ENaCs/ASICs had been reported to be associated with cancer characteristics. Given their cell surface localization and pharmacology, pharmacological strategies to target ENaC/ASIC family members may be promising cancer therapeutics.

Thermodynamic and conformational analysis of the interaction between antibody binding proteins and IgG.

PubMed

Tanwar, Neetu; Munde, Manoj

2018-06-01

Studying interaction of IgG with bacterial proteins such as proA (Protein A) and proG is essential for development in the areas of drug discovery and biotechnology. Some solution studies in the past have hinted at the possibility of variable binding ratios for IgG with proA and proG. Since earlier crystallographic studies focussed mostly on monomeric complexes, the knowledge about the binding interfaces and protein conformational changes involved in multimeric complexes is scarce. In this paper, we observed that single proA molecule was able to bind to three IgG molecules (1:3, proA:IgG) in ITC accentuating the presence of conformational flexibility in proA, corroborated also by CD results. By contrast, proG binds with 1:1 stoichiometry to IgG, which also involves key structural rearrangement within the binding interface of IgG-proG complex, confirmed by fluorescence KI quenching study. It is implicit from CD and fluorescence results that IgG does not undergo any significant conformational changes, which further suggests that proA and proG dictate the phenomenon of recognition in antibody complexes. ANS as a hydrophobic probe helped in revealing the distinctive antibody binding mechanism of proA and proG. Additionally, the binding competition experiments using ITC established that proA and proG cannot bind IgG concurrently. Copyright © 2018. Published by Elsevier B.V.

The cabABC Operon Essential for Biofilm and Rugose Colony Development in Vibrio vulnificus

PubMed Central

Park, Jin Hwan; Jo, Youmi; Jang, Song Yee; Kwon, Haenaem; Irie, Yasuhiko; Parsek, Matthew R.; Kim, Myung Hee; Choi, Sang Ho

2015-01-01

A transcriptome analysis identified Vibrio vulnificus cabABC genes which were preferentially expressed in biofilms. The cabABC genes were transcribed as a single operon. The cabA gene was induced by elevated 3′,5′-cyclic diguanylic acid (c-di-GMP) and encoded a calcium-binding protein CabA. Comparison of the biofilms produced by the cabA mutant and its parent strain JN111 in microtiter plates using crystal-violet staining demonstrated that CabA contributed to biofilm formation in a calcium-dependent manner under elevated c-di-GMP conditions. Genetic and biochemical analyses revealed that CabA was secreted to the cell exterior through functional CabB and CabC, distributed throughout the biofilm matrix, and produced as the biofilm matured. These results, together with the observation that CabA also contributes to the development of rugose colony morphology, indicated that CabA is a matrix-associated protein required for maturation, rather than adhesion involved in the initial attachment, of biofilms. Microscopic comparison of the structure of biofilms produced by JN111 and the cabA mutant demonstrated that CabA is an extracellular matrix component essential for the development of the mature biofilm structures in flow cells and on oyster shells. Exogenously providing purified CabA restored the biofilm- and rugose colony-forming abilities of the cabA mutant when calcium was available. Circular dichroism and size exclusion analyses revealed that calcium binding induces CabA conformational changes which may lead to multimerization. Extracellular complementation experiments revealed that CabA can assemble a functional matrix only when exopolysaccharides coexist. Consequently, the combined results suggested that CabA is a structural protein of the extracellular matrix and multimerizes to a conformation functional in building robust biofilms, which may render V. vulnificus to survive in hostile environments and reach a concentrated infective dose. PMID:26406498

Inflammasome Assembly in the Chorioamniotic Membranes during Spontaneous Labor at Term

PubMed Central

Gomez-Lopez, Nardhy; Romero, Roberto; Xu, Yi; Garcia-Flores, Valeria; Leng, Yaozhu; Panaitescu, Bogdan; Miller, Derek; Abrahams, Vikki M.; Hassan, Sonia S.

2017-01-01

Problem Inflammasome activation requires two steps: priming and assembly of the multimeric complex. The second step includes assembly of the sensor molecule and adaptor protein ASC (an apoptosis-associated speck-like protein containing a CARD), which results in ASC speck formation and the recruitment of caspase (CASP)-1. Herein, we investigated whether there is inflammasome assembly in the chorioamniotic membranes and choriodecidual leukocytes from women who underwent spontaneous labor at term. Method of Study Using in situ proximity ligation assays, ASC/CASP-1 complexes were determined in the chorioamniotic membranes from women who delivered at term without labor or underwent spontaneous labor at term with or without acute histologic chorioamnionitis (n=10–11 each). Also, ASC speck formation was determined by flow cytometry in the choriodecidual leukocytes isolated from women who delivered at term with or without spontaneous labor (n=9–12 each). Results 1) ASC/CASP-1 complexes were detected in the chorioamniotic membranes; 2) ASC/CASP-1 complexes were greater in the chorioamniotic membranes from women who underwent spontaneous labor at term than in those without labor; 3) ASC/CASP-1 complexes were even more abundant in the chorioamniotic membranes from women who underwent spontaneous labor at term with acute histologic chorioamnionitis than in those without this placental lesion; 4) ASC speck formation was detected in the choriodecidual leukocytes; and 5) ASC speck formation was greater in the choriodecidual leukocytes isolated from women who underwent spontaneous labor at term than in those without labor. Conclusion There is inflammasome assembly in the chorioamniotic membranes and choriodecidual leukocytes during spontaneous labor at term. PMID:28233423

The naked and the dead: the ABCs of gymnosperm reproduction and the origin of the angiosperm flower.

PubMed

Melzer, Rainer; Wang, Yong-Qiang; Theissen, Günter

2010-02-01

20 years after establishment of the ABC model many of the molecular mechanisms underlying development of the angiosperm flower are relatively well understood. Central players in the gene regulatory network controlling flower development are SQUA-like, DEF/GLO-like, AG-like and AGL6/SEP1-like MIKC-type MADS-domain transcription factors. These provide class A, class B, class C and the more recently defined class E floral homeotic functions, respectively. There is evidence that the floral homeotic proteins recognize the DNA of target genes in an organ-specific way as multimeric protein complexes, thus constituting 'floral quartets'. In contrast to the detailed insights into flower development, how the flower originated during evolution has remained enigmatic. However, while orthologues of all classes of floral homeotic genes appear to be absent from all non-seed plants, DEF/GLO-like, AG-like, and AGL6-like genes have been found in diverse extant gymnosperms, the closest relatives of the angiosperms. While SQUA-like and SEP1-like MADS-box genes appear to be absent from extant gymnosperms, reconstruction of MADS-box gene phylogeny surprisingly suggests that the most recent common ancestor of gymnosperms and angiosperms possessed representatives of both genes, but that these have been lost in the lineage that led to extant gymnosperms. Expression studies and genetic complementation experiments indicate that both angiosperm and gymnosperm AG-like and DEF/GLO-like genes have conserved functions in the specification of reproductive organs and in distinguishing male from female organs, respectively. Based on these findings novel models about the molecular basis of flower origin, involving changes in the expression patterns of DEF/GLO-like or AGL6/SEP1/SQUA-like genes in reproductive structures, were developed. While in angiosperms SEP1-like proteins play an important role in floral quartet formation, preliminary evidence suggests that gymnosperm DEF/GLO-like and AG-like proteins alone can already form floral quartet-like complexes, further corroborating the view that the formation of floral quartet-like complexes predated flower origin during evolution. Copyright 2009 Elsevier Ltd. All rights reserved.

Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97

PubMed Central

Sonntag, Eric; Wagner, Sabrina; Strojan, Hanife; Wangen, Christina; Lenac Rovis, Tihana; Lisnic, Berislav; Jonjic, Stipan; Schlötzer-Schrehardt, Ursula; Marschall, Manfred

2018-01-01

The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction. PMID:29342872

Excitation energy transfer in the photosystem I

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

Webber, Andrew N

2012-09-25

Photosystem I is a multimeric pigment protein complex in plants, green alage and cyanobacteria that functions in series with Photosystem II to use light energy to oxidize water and reduce carbon dioxide. The Photosystem I core complex contains 96 chlorophyll a molecules and 22 carotenoids that are involved in light harvesting and electron transfer. In eucaryotes, PSI also has a peripheral light harvesting complex I (LHCI). The role of specific chlorophylls in excitation and electron transfer are still unresolved. In particular, the role of so-called bridging chlorophylls, located between the bulk antenna and the core electron transfer chain, in themore » transfer of excitation energy to the reaction center are unknown. During the past funding period, site directed mutagenesis has been used to create mutants that effect the physical properties of these key chlorophylls, and to explore how this alters the function of the photosystem. Studying these mutants using ultrafast absorption spectroscopy has led to a better understanding of the process by which excitation energy is transferred from the antenna chlorophylls to the electron transfer chain chlorophylls, and what the role of connecting chlorophylls and A_0 chlorophylls is in this process. We have also used these mutants to investigate whch of the central group of six chlorophylls are involved in the primary steps of charge separation and electron transfer.« less

Examining Myddosome Formation by Luminescence-Based Mammalian Interactome Mapping (LUMIER).

PubMed

Wolz, Olaf-Oliver; Koegl, Manfred; Weber, Alexander N R

2018-01-01

Recent structural, biochemical, and functional studies have led to the notion that many of the post-receptor signaling complexes in innate immunity have a multimeric, multi-protein architecture whose hierarchical assembly is vital for function. The Myddosome is a post-receptor complex in the cytoplasmic signaling of Toll-like receptors (TLR) and the Interleukin-1 receptor (IL-1R), involving the proteins MyD88, IL-1R-associated kinase 4 (IRAK4), and IRAK2. Its importance is strikingly illustrated by the fact that rare germline mutations in MYD88 causing high susceptibility to infections are characterized by failure to assemble Myddosomes; conversely, gain-of-function MYD88 mutations leading to oncogenic hyperactivation of NF-κB show increased Myddosome formation. Reliable methods to probe Myddosome formation experimentally are therefore vital to further study the properties of this important post-receptor complex and its role in innate immunity, such as its regulation by posttranslational modification. Compared to structural and biochemical analyses, luminescence-based mammalian interactome mapping (LUMIER) is a straightforward, automatable, quantifiable, and versatile technique to study protein-protein interactions in a physiologically relevant context. We adapted LUMIER for Myddosome analysis and provide here a basic background of this technique, suitable experimental protocols, and its potential for medium-throughput screening. The principles presented herein can be adapted to other signaling pathways.

Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry as a Platform for Characterizing Multimeric Membrane Protein Complexes

NASA Astrophysics Data System (ADS)

Lippens, Jennifer L.; Nshanian, Michael; Spahr, Chris; Egea, Pascal F.; Loo, Joseph A.; Campuzano, Iain D. G.

2018-01-01

Membrane protein characterization is consistently hampered by challenges with expression, purification, and solubilization. Among several biophysical techniques employed for their characterization, native-mass spectrometry (MS) has emerged as a powerful tool for the analysis of membrane proteins and complexes. Here, two MS platforms, the FT-ICR and Q-ToF, have been explored to analyze the homotetrameric water channel protein, AquaporinZ (AqpZ), under non-denaturing conditions. This 97 kDa membrane protein complex can be readily liberated from the octylglucoside (OG) detergent micelle under a range of instrument conditions on both MS platforms. Increasing the applied collision energy of the FT-ICR collision cell yielded varying degrees of tetramer (97 kDa) liberation from the OG micelles, as well as dissociation into the trimeric (72 kDa) and monomeric (24 kDa) substituents. Tandem-MS on the Q-ToF yielded higher intensity tetramer signal and, depending on the m/z region selected, the observed monomer signal varied in intensity. Precursor ion selection of an m/z range above the expected protein signal distribution, followed by mild collisional activation, is able to efficiently liberate AqpZ with a high S/N ratio. The tetrameric charge state distribution obtained on both instruments demonstrated superpositioning of multiple proteoforms due to varying degrees of N-terminal formylation. [Figure not available: see fulltext.

Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability

PubMed Central

Choy, John S.; O'Toole, Eileen; Schuster, Breanna M.; Crisp, Matthew J.; Karpova, Tatiana S.; McNally, James G.; Winey, Mark; Gardner, Melissa K.; Basrai, Munira A.

2013-01-01

How subunit dosage contributes to the assembly and function of multimeric complexes is an important question with implications in understanding biochemical, evolutionary, and disease mechanisms. Toward identifying pathways that are susceptible to decreased gene dosage, we performed a genome-wide screen for haploinsufficient (HI) genes that guard against genome instability in Saccharomyces cerevisiae. This led to the identification of all three genes (SPC97, SPC98, and TUB4) encoding the evolutionarily conserved γ-tubulin small complex (γ-TuSC), which nucleates microtubule assembly. We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities. Fluorescence microscopy, fluorescence recovery after photobleaching, electron tomography, and model convolution simulation of spc98/+ mutants revealed improper regulation of interpolar (iMT) and kinetochore (kMT) microtubules in anaphase. The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants. We propose that γ-TuSC is crucial for balanced assembly between iMTs and kMTs for spindle organization and accurate chromosome segregation. Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles. PMID:23825022

Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability.

PubMed

Choy, John S; O'Toole, Eileen; Schuster, Breanna M; Crisp, Matthew J; Karpova, Tatiana S; McNally, James G; Winey, Mark; Gardner, Melissa K; Basrai, Munira A

2013-09-01

How subunit dosage contributes to the assembly and function of multimeric complexes is an important question with implications in understanding biochemical, evolutionary, and disease mechanisms. Toward identifying pathways that are susceptible to decreased gene dosage, we performed a genome-wide screen for haploinsufficient (HI) genes that guard against genome instability in Saccharomyces cerevisiae. This led to the identification of all three genes (SPC97, SPC98, and TUB4) encoding the evolutionarily conserved γ-tubulin small complex (γ-TuSC), which nucleates microtubule assembly. We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities. Fluorescence microscopy, fluorescence recovery after photobleaching, electron tomography, and model convolution simulation of spc98/+ mutants revealed improper regulation of interpolar (iMT) and kinetochore (kMT) microtubules in anaphase. The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants. We propose that γ-TuSC is crucial for balanced assembly between iMTs and kMTs for spindle organization and accurate chromosome segregation. Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles.

Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97.

PubMed

Milbradt, Jens; Sonntag, Eric; Wagner, Sabrina; Strojan, Hanife; Wangen, Christina; Lenac Rovis, Tihana; Lisnic, Berislav; Jonjic, Stipan; Sticht, Heinrich; Britt, William J; Schlötzer-Schrehardt, Ursula; Marschall, Manfred

2018-01-13

The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

pH dependence of the dissociation of multimeric hemoglobin probed by high hydrostatic pressure.

PubMed

Bispo, Jose A C; Santos, Jose L R; Landini, Gustavo F; Goncalves, Juliana M; Bonafe, Carlos F S

2007-02-01

We investigated the thermodynamic features of the classic alkaline dissociation of multimeric hemoglobin (3.1 MDa) from Glossoscolex paulistus (Annelidea) using high hydrostatic pressure. Light scattering measurements up to microscopic thermodynamic equilibrium indicated a high pH dependency of dissociation and association. Electron microscopy and gel filtration corroborated these findings. The volume change of dissociation decreased in absolute values from -48.0 mL/mol of subunit at pH 6.0 to -19.2 mL/mol at pH 9.0, suggesting a lack of protein interactions under alkaline conditions. Concomitantly, an increase in pH reduced the Gibbs free energy of dissociation from 37.7 to 27.5 kJ/mol of subunit. The stoichiometry of proton release calculated from the pressure-induced dissociation curves was +0.602 mol of H(+)/mol of subunit. These results provide a direct quantification of proton participation in stabilizing the aggregated state of the hemoglobin, and contribute to our understanding of protein-protein interactions and of the surrounding conditions that modulate the process of aggregation.

Enhancing immunogenicity and transmission-blocking activity of malaria vaccines by fusing Pfs25 to IMX313 multimerization technology.

PubMed

Li, Yuanyuan; Leneghan, Darren B; Miura, Kazutoyo; Nikolaeva, Daria; Brian, Iona J; Dicks, Matthew D J; Fyfe, Alex J; Zakutansky, Sarah E; de Cassan, Simone; Long, Carole A; Draper, Simon J; Hill, Adrian V S; Hill, Fergal; Biswas, Sumi

2016-01-08

Transmission-blocking vaccines (TBV) target the sexual-stages of the malaria parasite in the mosquito midgut and are widely considered to be an essential tool for malaria elimination. High-titer functional antibodies are required against target antigens to achieve effective transmission-blocking activity. We have fused Pfs25, the leading malaria TBV candidate antigen to IMX313, a molecular adjuvant and expressed it both in ChAd63 and MVA viral vectors and as a secreted protein-nanoparticle. Pfs25-IMX313 expressed from viral vectors or as a protein-nanoparticle is significantly more immunogenic and gives significantly better transmission-reducing activity than monomeric Pfs25. In addition, we demonstrate that the Pfs25-IMX313 protein-nanoparticle leads to a qualitatively improved antibody response in comparison to soluble Pfs25, as well as to significantly higher germinal centre (GC) responses. These results demonstrate that antigen multimerization using IMX313 is a very promising strategy to enhance antibody responses against Pfs25, and that Pfs25-IMX313 is a highly promising TBV candidate vaccine.

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

DOE PAGES

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

2015-04-09

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

A chemotactic signaling surface on CheY defined by suppressors of flagellar switch mutations.

PubMed Central

Roman, S J; Meyers, M; Volz, K; Matsumura, P

1992-01-01

CheY is the response regulator protein that interacts with the flagellar switch apparatus to modulate flagellar rotation during chemotactic signaling. CheY can be phosphorylated and dephosphorylated in vitro, and evidence indicates that CheY-P is the activated form that induces clockwise flagellar rotation, resulting in a tumble in the cell's swimming pattern. The flagellar switch apparatus is a complex macromolecular structure composed of at least three gene products, FliG, FliM, and FliN. Genetic analysis of Escherichia coli has identified fliG and fliM as genes in which mutations occur that allele specifically suppress cheY mutations, indicating interactions among these gene products. We have generated a class of cheY mutations selected for dominant suppression of fliG mutations. Interestingly, these cheY mutations dominantly suppressed both fliG and fliM mutations; this is consistent with the idea that the CheY protein interacts with both switch gene products during signaling. Biochemical characterization of wild-type and suppressor CheY proteins did not reveal altered phosphorylation properties or evidence for phosphorylation-dependent CheY multimerization. These data indicate that suppressor CheY proteins are specifically altered in the ability to transduce chemotactic signals to the switch at some point subsequent to phosphorylation. Physical mapping of suppressor amino acid substitutions on the crystal structure of CheY revealed a high degree of spatial clustering, suggesting that this region of CheY is a signaling surface that transduces chemotactic signals to the switch. Images PMID:1400175

Adaptability of Protein Structures to Enable Functional Interactions and Evolutionary Implications

PubMed Central

Haliloglu, Turkan; Bahar, Ivet

2015-01-01

Several studies in recent years have drawn attention to the ability of proteins to adapt to intermolecular interactions by conformational changes along structure-encoded collective modes of motions. These so-called soft modes, primarily driven by entropic effects, facilitate, if not enable, functional interactions. They represent excursions on the conformational space along principal low-ascent directions/paths away from the original free energy minimum, and they are accessible to the protein even prior to protein-protein/ligand interactions. An emerging concept from these studies is the evolution of structures or modular domains to favor such modes of motion that will be recruited or integrated for enabling functional interactions. Structural dynamics, including the allosteric switches in conformation that are often stabilized upon formation of complexes and multimeric assemblies, emerge as key properties that are evolutionarily maintained to accomplish biological activities, consistent with the paradigm sequence → structure → dynamics → function where ‘dynamics’ bridges structure and function. PMID:26254902

Peptide-MHC-based nanomedicines for autoimmunity function as T-cell receptor microclustering devices

NASA Astrophysics Data System (ADS)

Singha, Santiswarup; Shao, Kun; Yang, Yang; Clemente-Casares, Xavier; Solé, Patricia; Clemente, Antonio; Blanco, Jesús; Dai, Qin; Song, Fayi; Liu, Shang Wan; Yamanouchi, Jun; Umeshappa, Channakeshava Sokke; Nanjundappa, Roopa Hebbandi; Detampel, Pascal; Amrein, Matthias; Fandos, César; Tanguay, Robert; Newbigging, Susan; Serra, Pau; Khadra, Anmar; Chan, Warren C. W.; Santamaria, Pere

2017-07-01

We have shown that nanoparticles (NPs) can be used as ligand-multimerization platforms to activate specific cellular receptors in vivo. Nanoparticles coated with autoimmune disease-relevant peptide-major histocompatibility complexes (pMHC) blunted autoimmune responses by triggering the differentiation and expansion of antigen-specific regulatory T cells in vivo. Here, we define the engineering principles impacting biological activity, detail a synthesis process yielding safe and stable compounds, and visualize how these nanomedicines interact with cognate T cells. We find that the triggering properties of pMHC-NPs are a function of pMHC intermolecular distance and involve the sustained assembly of large antigen receptor microclusters on murine and human cognate T cells. These compounds show no off-target toxicity in zebrafish embryos, do not cause haematological, biochemical or histological abnormalities, and are rapidly captured by phagocytes or processed by the hepatobiliary system. This work lays the groundwork for the design of ligand-based NP formulations to re-program in vivo cellular responses using nanotechnology.

Adapting to stress - chaperome networks in cancer.

PubMed

Joshi, Suhasini; Wang, Tai; Araujo, Thaís L S; Sharma, Sahil; Brodsky, Jeffrey L; Chiosis, Gabriela

2018-05-23

In this Opinion article, we aim to address how cells adapt to stress and the repercussions chronic stress has on cellular function. We consider acute and chronic stress-induced changes at the cellular level, with a focus on a regulator of cellular stress, the chaperome, which is a protein assembly that encompasses molecular chaperones, co-chaperones and other co-factors. We discuss how the chaperome takes on distinct functions under conditions of stress that are executed in ways that differ from the one-on-one cyclic, dynamic functions exhibited by distinct molecular chaperones. We argue that through the formation of multimeric stable chaperome complexes, a state of chaperome hyperconnectivity, or networking, is gained. The role of these chaperome networks is to act as multimolecular scaffolds, a particularly important function in cancer, where they increase the efficacy and functional diversity of several cellular processes. We predict that these concepts will change how we develop and implement drugs targeting the chaperome to treat cancer.

Bioengineered protein-based nanocage for drug delivery.

PubMed

Lee, Eun Jung; Lee, Na Kyeong; Kim, In-San

2016-11-15

Nature, in its wonders, presents and assembles the most intricate and delicate protein structures and this remarkable phenomenon occurs in all kingdom and phyla of life. Of these proteins, cage-like multimeric proteins provide spatial control to biological processes and also compartmentalizes compounds that may be toxic or unstable and avoids their contact with the environment. Protein-based nanocages are of particular interest because of their potential applicability as drug delivery carriers and their perfect and complex symmetry and ideal physical properties, which have stimulated researchers to engineer, modify or mimic these qualities. This article reviews various existing types of protein-based nanocages that are used for therapeutic purposes, and outlines their drug-loading mechanisms and bioengineering strategies via genetic and chemical functionalization. Through a critical evaluation of recent advances in protein nanocage-based drug delivery in vitro and in vivo, an outlook for de novo and in silico nanocage design, and also protein-based nanocage preclinical and future clinical applications will be presented. Copyright © 2016 Elsevier B.V. All rights reserved.

The Competitive Interplay between Allosteric HIV-1 Integrase Inhibitor BI/D and LEDGF/p75 during the Early Stage of HIV-1 Replication Adversely Affects Inhibitor Potency.

PubMed

Feng, Lei; Dharmarajan, Venkatasubramanian; Serrao, Erik; Hoyte, Ashley; Larue, Ross C; Slaughter, Alison; Sharma, Amit; Plumb, Matthew R; Kessl, Jacques J; Fuchs, James R; Bushman, Frederic D; Engelman, Alan N; Griffin, Patrick R; Kvaratskhelia, Mamuka

2016-05-20

Allosteric HIV-1 integrase inhibitors (ALLINIs) have recently emerged as a promising class of antiretroviral agents and are currently in clinical trials. In infected cells, ALLINIs potently inhibit viral replication by impairing virus particle maturation but surprisingly exhibit a reduced EC50 for inhibiting HIV-1 integration in target cells. To better understand the reduced antiviral activity of ALLINIs during the early stage of HIV-1 replication, we investigated the competitive interplay between a potent representative ALLINI, BI/D, and LEDGF/p75 with HIV-1 integrase. While the principal binding sites of BI/D and LEDGF/p75 overlap at the integrase catalytic core domain dimer interface, we show that the inhibitor and the cellular cofactor induce markedly different multimerization patterns of full-length integrase. LEDGF/p75 stabilizes an integrase tetramer through the additional interactions with the integrase N-terminal domain, whereas BI/D induces protein-protein interactions in C-terminal segments that lead to aberrant, higher-order integrase multimerization. We demonstrate that LEDGF/p75 binds HIV-1 integrase with significantly higher affinity than BI/D and that the cellular protein is able to reverse the inhibitor induced aberrant, higher-order integrase multimerization in a dose-dependent manner in vitro. Consistent with these observations, alterations of the cellular levels of LEDGF/p75 markedly affected BI/D EC50 values during the early steps of HIV-1 replication. Furthermore, genome-wide sequencing of HIV-1 integration sites in infected cells demonstrate that LEDGF/p75-dependent integration site selection is adversely affected by BI/D treatment. Taken together, our studies elucidate structural and mechanistic details of the interplay between LEDGF/p75 and BI/D during the early stage of HIV-1 replication.

Vaccination with multimeric recombinant VP28 induces high protection against white spot syndrome virus in shrimp.

PubMed

Taengchaiyaphum, Suparat; Nakayama, Hideki; Srisala, Jiraporn; Khiev, Ratny; Aldama-Cano, Diva January; Thitamadee, Siripong; Sritunyalucksana, Kallaya

2017-11-01

To improve the efficacy of WSSV protection, multimeric (tetrameric) recombinant VP28 (4XrVP28) was produced and tested in comparison with those of monomeric VP28 (1XrVP28). In vitro binding of either 1XrVP28 or 4XrVP28 to shrimp hemocyte surface was evident as early as 10 min after protein inoculation. Similar results were obtained in vivo when shrimp were injected with recombinant proteins that the proteins bound to the hemocyte surface could be detected since 5 min after injection. Comparison of the WSSV protection efficiencies of 1XrVP28 or 4XrVP28 were performed by injection the purified 1XrVP28 or 4XrVP28 (22.5 μg/shrimp) and WSSV inoculum (1000 copies/shrimp) into shrimp. At 10 dpi, while shrimp injected with WSSV inoculum reached 100% mortality, shrimp injected with 1XrVP28 + WSSV or 4XrVP28 + WSSV showed relative percent survival (RPS) of 67% and 81%, respectively. PCR quantification revealed high number of WSSV in the moribund shrimp of WSSV- and 1XrVP28+WSSV-injected group. In contrast, lower number of WSSV copies were found in the survivors both from 1XrVP28+WSSV- or 4XrVP28+WSSV- injected groups. Histopathological analysis demonstrated the WSSV infected lesions found in the moribund from WSSV-infected group and 1XrVP28+WSSV-injected group, but less or none in the survivors. ELISA demonstrated that 4XrVP28 exhibited higher affinity binding to rPmRab7, a WSSV binding protein essential for WSSV entry to the cell than 1XrVP28. Taken together, the protection against WSSV in shrimp could be improved by application of multimeric rVP28. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparative analysis of von Willebrand factor profiles after implantation of left ventricular assist device and total artificial heart.

PubMed

Reich, H J; Morgan, J; Arabia, F; Czer, L; Moriguchi, J; Ramzy, D; Esmailian, F; Lam, L; Dunhill, J; Volod, O

2017-08-01

Essentials Bleeding is a major source of morbidity during mechanical circulatory support. von Willebrand factor (VWF) multimer loss may contribute to bleeding. Different patterns of VWF multimer loss were seen with the two device types. This is the first report of total artificial heart associated VWF multimer loss. Background Bleeding remains a challenge during mechanical circulatory support and underlying mechanisms are incompletely understood. Functional von Willebrand factor (VWF) impairment because of loss of high-molecular-weight multimers (MWMs) produces acquired von Willebrand disease (VWD) after left ventricular assist device (LVAD). Little is known about VWF multimers with total artificial hearts (TAHs). Here, VWF profiles with LVADs and TAHs are compared using a VWD panel. Methods VWD evaluations for patients with LVAD or TAH (2013-14) were retrospectively analyzed and included: VWF activity (ristocetin cofactor, VWF:RCo), VWF antigen (VWF:Ag), ratio of VWF:RCo to VWF:Ag, and quantitative VWF multimeric analysis. Results Twelve patients with LVADs and 12 with TAHs underwent VWD evaluation. All had either normal (47.8%) or elevated (52.2%) VWF:RCo, normal (26.1%) or elevated (73.9%) VWF:Ag and 50.0% were disproportional (ratio ≤ 0.7). Multimeric analysis showed abnormal patterns in all patients with LVADs: seven with high MWM loss; five with highest MWM loss. With TAH, 10/12 patients had abnormal patterns: all with highest MWM loss. High MWM loss correlated with presence of LVAD and highest MWM loss with TAH. Increased low MWMs were detected in 22/24. Conclusion Using VWF multimeric analysis, abnormalities after LVAD or TAH were detected that would be missed with measurements of VWF level alone: loss of high MWM predominantly in LVAD, loss of highest MWM in TAH, and elevated levels of low MWM in both. This is the first study to describe TAH-associated highest MWM loss, which may contribute to bleeding. © 2017 International Society on Thrombosis and Haemostasis.

A mutation within the transmembrane domain of melanosomal protein Silver (Pmel17) changes lumenal fragment interactions

PubMed Central

Kuliawat, Regina; Santambrogio, Laura

2009-01-01

Melanocytes synthesize and store melanin within tissue-specific organelles, the melanosomes. Melanin deposition takes place along fibrils found within these organelles and fibril formation is known to depend on trafficking of the membrane glycoprotein Silver/Pmel17. However, correctly targeted, full-length Silver/Pmel17 cannot form fibers. Proteolytic processing in endosomal compartments and the generation of a lumenal Mα fragment that is incorporated into amyloid-like structures is also essential. Dominant White (DWhite), a mutant form of Silver/Pmel17 first described in chicken, causes disorganized fibers and severe hypopigmentation due to melanocyte death. Surprisingly, the DWhite mutation is an insertion of three amino acids into the transmembrane domain; the DWhite-Mα fragment is unaffected. To determine the functional importance of the transmembrane domain in organized fibril assembly, we investigated membrane trafficking and multimerization of Silver/Pmel17/DWhite proteins. We demonstrate that the DWhite mutation changes lipid interactions and disulfide bond-mediated associations of lumenal domains. Thus, partitioning into membrane microdomains and effects on conformation explain how the transmembrane region may contribute to the structural integrity of Silver/Pmel17 oligomers or influence toxic, amyloidogenic properties. PMID:19679373

Assembly of the Type II Secretion System such as Found in Vibrio cholerae Depends on the Novel Pilotin AspS

PubMed Central

Dunstan, Rhys A.; Heinz, Eva; Wijeyewickrema, Lakshmi C.; Pike, Robert N.; Purcell, Anthony W.; Evans, Timothy J.; Praszkier, Judyta; Robins-Browne, Roy M.; Strugnell, Richard A.; Korotkov, Konstantin V.; Lithgow, Trevor

2013-01-01

The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS. PMID:23326233

Antiviral RNA Recognition and Assembly by RLR Family Innate Immune Sensors

PubMed Central

Bruns, Annie M.; Horvath, Curt M.

2014-01-01

Virus-encoded molecular signatures, such as cytosolic double-stranded or otherwise biochemically distinct RNA species, trigger cellular antiviral signaling. Cytoplasmic proteins recognize these non-self RNAs and activate signal transduction pathways that drive the expression of virus-induced genes, including the primary antiviral cytokine, IFNβ, and diverse direct and indirect antiviral effectors [1–4]. One important group of cytosolic RNA sensors known as the RIG-I like receptors (RLRs) is comprised of three proteins that are similar in structure and function. The RLR proteins, RIG-I, MDA5, and LGP2, share the ability to recognize nucleic acid signatures produced by virus infections and activate antiviral signaling. Emerging evidence indicates that RNA detection by RLRs culminates in the assembly of dynamic multimeric ribonucleoprotein (RNP) complexes. These RNPs can act as signaling platforms that are capable of propagating and amplifying antiviral signaling responses. Despite their common domain structures and similar abilities to induce antiviral responses, the RLRs differ in their enzymatic properties, their intrinsic abilities to recognize RNA, and their ability to assemble into filamentous complexes. This molecular specialization has enabled the RLRs to recognize and respond to diverse virus infections, and to mediate both unique and overlapping functions in immune regulation [5, 6]. PMID:25081315

Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome

PubMed Central

ORBAN, TAMAS I.; IZAURRALDE, ELISA

2005-01-01

RNA interference (RNAi) is a conserved RNA silencing pathway that leads to sequence-specific mRNA decay in response to the presence of double-stranded RNA (dsRNA). Long dsRNA molecules are first processed by Dicer into 21–22-nucleotide small interfering RNAs (siRNAs). The siRNAs are incorporated into a multimeric RNA-induced silencing complex (RISC) that cleaves mRNAs at a site determined by complementarity with the siRNAs. Following this initial endonucleolytic cleavage, the mRNA is degraded by a mechanism that is not completely understood. We investigated the decay pathway of mRNAs targeted by RISC in Drosophila cells. We show that 5′ mRNA fragments generated by RISC cleavage are rapidly degraded from their 3′ ends by the exosome, whereas the 3′ fragments are degraded from their 5′ ends by XRN1. Exosome-mediated decay of the 5′ fragments requires the Drosophila homologs of yeast Ski2p, Ski3p, and Ski8p, suggesting that their role as regulators of exosome activity is conserved. Our findings indicate that mRNAs targeted by siRNAs are degraded from the ends generated by RISC cleavage, without undergoing decapping or deadenylation. PMID:15703439

Succinate dehydrogenase (SDHx) mutations in pituitary tumors: could this be a new role for mitochondrial complex II and/or Krebs cycle defects?

PubMed

Xekouki, Paraskevi; Stratakis, Constantine A

2012-12-01

Succinate dehydrogenase (SDH) or mitochondrial complex II is a multimeric enzyme that is bound to the inner membrane of mitochondria and has a dual role as it serves both as a critical step of the tricarboxylic acid or Krebs cycle and as a member of the respiratory chain that transfers electrons directly to the ubiquinone pool. Mutations in SDH subunits have been implicated in the formation of familial paragangliomas (PGLs) and/or pheochromocytomas (PHEOs) and in Carney-Stratakis syndrome. More recently, SDH defects were associated with predisposition to a Cowden disease phenotype, renal, and thyroid cancer. We recently described a kindred with the coexistence of familial PGLs and an aggressive GH-secreting pituitary adenoma, harboring an SDHD mutation. The pituitary tumor showed loss of heterozygosity at the SDHD locus, indicating the possibility that SDHD's loss was causatively linked to the development of the neoplasm. In total, 29 cases of pituitary adenomas presenting in association with PHEOs and/or extra-adrenal PGLs have been reported in the literature since 1952. Although a number of other genetic defects are possible in these cases, we speculate that the association of PHEOs and/or PGLs with pituitary tumors is a new syndromic association and a novel phenotype for SDH defects.

Succinate dehydrogenase (SDHx) mutations in pituitary tumors: could this be a new role for mitochondrial complex II and/or Krebs cycle defects?

PubMed Central

Xekouki, Paraskevi; Stratakis, Constantine A

2013-01-01

Succinate dehydrogenase (SDH) or mitochondrial complex II is a multimeric enzyme that is bound to the inner membrane of mitochondria and has a dual role as it serves both as a critical step of the tricarboxylic acid or Krebs cycle and as a member of the respiratory chain that transfers electrons directly to the ubiquinone pool. Mutations in SDH subunits have been implicated in the formation of familial paragangliomas (PGLs) and/or pheochromocytomas (PHEOs) and in Carney–Stratakis syndrome. More recently, SDH defects were associated with predisposition to a Cowden disease phenotype, renal, and thyroid cancer. We recently described a kindred with the coexistence of familial PGLs and an aggressive GH-secreting pituitary adenoma, harboring an SDHD mutation. The pituitary tumor showed loss of heterozygosity at the SDHD locus, indicating the possibility that SDHD’s loss was causatively linked to the development of the neoplasm. In total, 29 cases of pituitary adenomas presenting in association with PHEOs and/or extra-adrenal PGLs have been reported in the literature since 1952. Although a number of other genetic defects are possible in these cases, we speculate that the association of PHEOs and/or PGLs with pituitary tumors is a new syndromic association and a novel phenotype for SDH defects. PMID:22889736

Structure of the apoptosome: mechanistic insights into activation of an initiator caspase from Drosophila

PubMed Central

Pang, Yuxuan; Bai, Xiao-chen; Yan, Chuangye; Hao, Qi; Chen, Zheqin; Wang, Jia-Wei

2015-01-01

Apoptosis is executed by a cascade of caspase activation. The autocatalytic activation of an initiator caspase, exemplified by caspase-9 in mammals or its ortholog, Dronc, in fruit flies, is facilitated by a multimeric adaptor complex known as the apoptosome. The underlying mechanism by which caspase-9 or Dronc is activated by the apoptosome remains unknown. Here we report the electron cryomicroscopic (cryo-EM) structure of the intact apoptosome from Drosophila melanogaster at 4.0 Å resolution. Analysis of the Drosophila apoptosome, which comprises 16 molecules of the Dark protein (Apaf-1 ortholog), reveals molecular determinants that support the assembly of the 2.5-MDa complex. In the absence of dATP or ATP, Dronc zymogen potently induces formation of the Dark apoptosome, within which Dronc is efficiently activated. At 4.1 Å resolution, the cryo-EM structure of the Dark apoptosome bound to the caspase recruitment domain (CARD) of Dronc (Dronc-CARD) reveals two stacked rings of Dronc-CARD that are sandwiched between two octameric rings of the Dark protein. The specific interactions between Dronc-CARD and both the CARD and the WD40 repeats of a nearby Dark protomer are indispensable for Dronc activation. These findings reveal important mechanistic insights into the activation of initiator caspase by the apoptosome. PMID:25644603

Inflammasome assembly in the chorioamniotic membranes during spontaneous labor at term.

PubMed

Gomez-Lopez, Nardhy; Romero, Roberto; Xu, Yi; Garcia-Flores, Valeria; Leng, Yaozhu; Panaitescu, Bogdan; Miller, Derek; Abrahams, Vikki M; Hassan, Sonia S

2017-05-01

Inflammasome activation requires two steps: priming and assembly of the multimeric complex. The second step includes assembly of the sensor molecule and adaptor protein ASC (an apoptosis-associated speck-like protein containing a CARD), which results in ASC speck formation and the recruitment of caspase (CASP)-1. Herein, we investigated whether there is inflammasome assembly in the chorioamniotic membranes and choriodecidual leukocytes from women who underwent spontaneous labor at term. Using in situ proximity ligation assays, ASC/CASP-1 complexes were determined in the chorioamniotic membranes from women who delivered at term without labor or underwent spontaneous labor at term with or without acute histologic chorioamnionitis (n=10-11 each). Also, ASC speck formation was determined by flow cytometry in the choriodecidual leukocytes isolated from women who delivered at term with or without spontaneous labor (n=9-12 each). (i) ASC/CASP-1 complexes were detected in the chorioamniotic membranes; (ii) ASC/CASP-1 complexes were greater in the chorioamniotic membranes from women who underwent spontaneous labor at term than in those without labor; (iii) ASC/CASP-1 complexes were even more abundant in the chorioamniotic membranes from women who underwent spontaneous labor at term with acute histologic chorioamnionitis than in those without this placental lesion; (iv) ASC speck formation was detected in the choriodecidual leukocytes; and (v) ASC speck formation was greater in the choriodecidual leukocytes isolated from women who underwent spontaneous labor at term than in those without labor. There is inflammasome assembly in the chorioamniotic membranes and choriodecidual leukocytes during spontaneous labor at term. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting.

PubMed

Herbert, Kristina M; Sarkar, Susanta K; Mills, Maria; Delgado De la Herran, Hilda C; Neuman, Keir C; Steitz, Joan A

2016-02-01

During microRNA (miRNA) biogenesis, the Microprocessor complex (MC), composed minimally of Drosha, an RNaseIII enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary-miRNA (pri-miRNA) to release the pre-miRNA stem-loop structure. Size-exclusion chromatography of the MC, isolated from mammalian cells, suggested multiple copies of one or both proteins in the complex. However, the exact stoichiometry was unknown. Initial experiments suggested that DGCR8 bound pri-miRNA substrates specifically, and given that Drosha could not be bound or cross-linked to RNA, a sequential model for binding was established in which DGCR8 bound first and recruited Drosha. Therefore, many laboratories have studied DGCR8 binding to RNA in the absence of Drosha and have shown that deletion constructs of DGCR8 can multimerize in the presence of RNA. More recently, it was demonstrated that Drosha can bind pri-miRNA substrates in the absence of DGCR8, casting doubt on the sequential model of binding. In the same study, using a single-molecule photobleaching assay, fluorescent protein-tagged deletion constructs of DGCR8 and Drosha assembled into a heterotrimeric complex on RNA, comprising two DGCR8 molecules and one Drosha molecule. To determine the stoichiometry of Drosha and DGCR8 within the MC in the absence of added RNA, we also used a single-molecule photobleaching assay and confirmed the heterotrimeric model of the human MC. We demonstrate that a heterotrimeric complex is likely preformed in the absence of RNA and exists even when full-length proteins are expressed and purified from human cells, and when hAGT-derived tags are used rather than fluorescent proteins. © 2016 Herbert et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Bioluminescence resonance energy transfer system for measuring dynamic protein-protein interactions in bacteria.

PubMed

Cui, Boyu; Wang, Yao; Song, Yunhong; Wang, Tietao; Li, Changfu; Wei, Yahong; Luo, Zhao-Qing; Shen, Xihui

2014-05-20

Protein-protein interactions are important for virtually every biological process, and a number of elegant approaches have been designed to detect and evaluate such interactions. However, few of these methods allow the detection of dynamic and real-time protein-protein interactions in bacteria. Here we describe a bioluminescence resonance energy transfer (BRET) system based on the bacterial luciferase LuxAB. We found that enhanced yellow fluorescent protein (eYFP) accepts the emission from LuxAB and emits yellow fluorescence. Importantly, BRET occurred when LuxAB and eYFP were fused, respectively, to the interacting protein pair FlgM and FliA. Furthermore, we observed sirolimus (i.e., rapamycin)-inducible interactions between FRB and FKBP12 and a dose-dependent abolishment of such interactions by FK506, the ligand of FKBP12. Using this system, we showed that osmotic stress or low pH efficiently induced multimerization of the regulatory protein OmpR and that the multimerization induced by low pH can be reversed by a neutralizing agent, further indicating the usefulness of this system in the measurement of dynamic interactions. This method can be adapted to analyze dynamic protein-protein interactions and the importance of such interactions in bacterial processes such as development and pathogenicity. Real-time measurement of protein-protein interactions in prokaryotes is highly desirable for determining the roles of protein complex in the development or virulence of bacteria, but methods that allow such measurement are not available. Here we describe the development of a bioluminescence resonance energy transfer (BRET) technology that meets this need. The use of endogenous excitation light in this strategy circumvents the requirement for the sophisticated instrument demanded by standard fluorescence resonance energy transfer (FRET). Furthermore, because the LuxAB substrate decanal is membrane permeable, the assay can be performed without lysing the bacterial cells, thus allowing the detection of protein-protein interactions in live bacterial cells. This BRET system added another useful tool to address important questions in microbiological studies. Copyright © 2014 Cui et al.

Regulated transport into the nucleus of herpesviridae DNA replication core proteins.

PubMed

Gualtiero, Alvisi; Jans, David A; Camozzi, Daria; Avanzi, Simone; Loregian, Arianna; Ripalti, Alessandro; Palù, Giorgio

2013-09-16

The Herpesvirdae family comprises several major human pathogens belonging to three distinct subfamilies. Their double stranded DNA genome is replicated in the nuclei of infected cells by a number of host and viral products. Among the latter the viral replication complex, whose activity is strictly required for viral replication, is composed of six different polypeptides, including a two-subunit DNA polymerase holoenzyme, a trimeric primase/helicase complex and a single stranded DNA binding protein. The study of herpesviral DNA replication machinery is extremely important, both because it provides an excellent model to understand processes related to eukaryotic DNA replication and it has important implications for the development of highly needed antiviral agents. Even though all known herpesviruses utilize very similar mechanisms for amplification of their genomes, the nuclear import of the replication complex components appears to be a heterogeneous and highly regulated process to ensure the correct spatiotemporal localization of each protein. The nuclear transport process of these enzymes is controlled by three mechanisms, typifying the main processes through which protein nuclear import is generally regulated in eukaryotic cells. These include cargo post-translational modification-based recognition by the intracellular transporters, piggy-back events allowing coordinated nuclear import of multimeric holoenzymes, and chaperone-assisted nuclear import of specific subunits. In this review we summarize these mechanisms and discuss potential implications for the development of antiviral compounds aimed at inhibiting the Herpesvirus life cycle by targeting nuclear import of the Herpesvirus DNA replicating enzymes.

SdAb heterodimer formation using leucine zippers

NASA Astrophysics Data System (ADS)

Goldman, Ellen R.; Anderson, George P.; Brozozog-Lee, P. Audrey; Zabetakis, Dan

2013-05-01

Single domain antibodies (sdAb) are variable domains cloned from camel, llama, or shark heavy chain only antibodies, and are among the smallest known naturally derived antigen binding fragments. SdAb derived from immunized llamas are able to bind antigens with high affinity, and most are capable of refolding after heat or chemical denaturation to bind antigen again. We hypothesized that the ability to produce heterodimeric sdAb would enable reagents with the robust characteristics of component sdAb, but with dramatically improved overall affinity through increased avidity. Previously we had constructed multimeric sdAb by genetically linking sdAb that bind non-overlapping epitopes on the toxin, ricin. In this work we explored a more flexible approach; the construction of multivalent binding reagents using multimerization domains. We expressed anti-ricin sdAb that recognize different epitopes on the toxin as fusions with differently charged leucine zippers. When the initially produced homodimers are mixed the leucine zipper domains will pair to produce heterodimers. We used fluorescence resonance energy transfer to confirm heterodimer formation. Surface plasmon resonance, circular dichroism, enzyme linked immunosorbent assays, and fluid array assays were used to characterize the multimer constructs, and evaluate their utility in toxin detection.

Influence of Oxidation and Multimerization on the Immunogenicity of a Thioredoxin-L2 Prophylactic Papillomavirus Vaccine

PubMed Central

Seitz, Hanna; Dantheny, Tatiana; Burkart, Frank; Ottonello, Simone

2013-01-01

Current commercial prophylactic human papillomavirus (HPV) vaccines are based on virus-like particles assembled from the major capsid protein L1 and show excellent safety and efficacy profiles. Still, a major limitation is their rather narrow range of protection against different HPV types. In contrast, the minor capsid protein L2 contains a so-called major cross-neutralizing epitope that can induce broad-range protective responses against multiple HPV types. This epitope is conserved among different papillomaviruses (PV) and contains two cysteine residues that are present in the L2 proteins of all known PV types. The main challenge in developing L2-directed vaccines is to overcome the intrinsically low immunogenicity of the L2 protein. Previously, we developed a recombinant L2-based prototype vaccine by inserting peptide epitopes spanning the cross-neutralizing L2 sequence into a bacterial thioredoxin (Trx) scaffold. These antigens induced high-titer neutralizing antibodies in mice. Here, we address the question of whether Trx scaffold multimerization may further enhance the immunogenicity of the TrxL2 vaccine. We also demonstrate that the oxidation state of the conserved cysteine residues is not essential for vaccine functionality, but it contributes to immunogenicity. PMID:23677323

The Centrosome-Specific Phosphorylation of Cnn by Polo/Plk1 Drives Cnn Scaffold Assembly and Centrosome Maturation

PubMed Central

Conduit, Paul T.; Feng, Zhe; Richens, Jennifer H.; Baumbach, Janina; Wainman, Alan; Bakshi, Suruchi D.; Dobbelaere, Jeroen; Johnson, Steven; Lea, Susan M.; Raff, Jordan W.

2014-01-01

Summary Centrosomes are important cell organizers. They consist of a pair of centrioles surrounded by pericentriolar material (PCM) that expands dramatically during mitosis—a process termed centrosome maturation. How centrosomes mature remains mysterious. Here, we identify a domain in Drosophila Cnn that appears to be phosphorylated by Polo/Plk1 specifically at centrosomes during mitosis. The phosphorylation promotes the assembly of a Cnn scaffold around the centrioles that is in constant flux, with Cnn molecules recruited continuously around the centrioles as the scaffold spreads slowly outward. Mutations that block Cnn phosphorylation strongly inhibit scaffold assembly and centrosome maturation, whereas phosphomimicking mutations allow Cnn to multimerize in vitro and to spontaneously form cytoplasmic scaffolds in vivo that organize microtubules independently of centrosomes. We conclude that Polo/Plk1 initiates the phosphorylation-dependent assembly of a Cnn scaffold around centrioles that is essential for efficient centrosome maturation in flies. PMID:24656740

The centrosome-specific phosphorylation of Cnn by Polo/Plk1 drives Cnn scaffold assembly and centrosome maturation.

PubMed

Conduit, Paul T; Feng, Zhe; Richens, Jennifer H; Baumbach, Janina; Wainman, Alan; Bakshi, Suruchi D; Dobbelaere, Jeroen; Johnson, Steven; Lea, Susan M; Raff, Jordan W

2014-03-31

Centrosomes are important cell organizers. They consist of a pair of centrioles surrounded by pericentriolar material (PCM) that expands dramatically during mitosis-a process termed centrosome maturation. How centrosomes mature remains mysterious. Here, we identify a domain in Drosophila Cnn that appears to be phosphorylated by Polo/Plk1 specifically at centrosomes during mitosis. The phosphorylation promotes the assembly of a Cnn scaffold around the centrioles that is in constant flux, with Cnn molecules recruited continuously around the centrioles as the scaffold spreads slowly outward. Mutations that block Cnn phosphorylation strongly inhibit scaffold assembly and centrosome maturation, whereas phosphomimicking mutations allow Cnn to multimerize in vitro and to spontaneously form cytoplasmic scaffolds in vivo that organize microtubules independently of centrosomes. We conclude that Polo/Plk1 initiates the phosphorylation-dependent assembly of a Cnn scaffold around centrioles that is essential for efficient centrosome maturation in flies. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Antibody reactivity to the major fish allergen parvalbumin is determined by isoforms and impact of thermal processing.

PubMed

Saptarshi, Shruti R; Sharp, Michael F; Kamath, Sandip D; Lopata, Andreas L

2014-04-01

The EF-hand calcium binding protein, parvalbumin, is a major fish allergen. Detection of this allergen is often difficult due to its structural diversity among various fish species. The aim of this study was to evaluate the cross-reactivity of parvalbumin in a comprehensive range of bony and cartilaginous fish, from the Asia-Pacific region, and conduct a molecular analysis of this highly allergenic protein. Using the monoclonal anti-parvalbumin antibody PARV-19, we demonstrated the presence of monomeric and oligomeric parvalbumin in all fish analysed, except for gummy shark a cartilaginous fish. Heat processing of this allergen greatly affected its antibody reactivity. While heating caused a reduction in antibody reactivity to multimeric forms of parvalbumins for most bony fish, a complete loss of reactivity was observed for cartilaginous fish. Molecular analysis demonstrated that parvalbumin cross-reactivity, among fish species, is due to the molecular phylogenetic association of this major fish allergen. Copyright © 2013 Elsevier Ltd. All rights reserved.

The Thiamin Pyrophosphate-Motif

NASA Technical Reports Server (NTRS)

Dominiak, Paulina M.; Ciszak, Ewa M.

2003-01-01

Using databases the authors have identified a common thiamin pyrophosphate (TPP)-motif in the family of functionally diverse TPP-dependent enzymes. This common motif consists of multimeric organization of subunits, two catalytic centers, common amino acid sequence, and specific contacts to provide a flip-flop, or alternate site, mechanism of action. Each catalytic center [PP:PYR] is formed at the interface of the PP-domain binding the magnesium ion, pyrophosphate and aminopyrimidine ring of TPP, and the PYR-domain binding the aminopyrimidine ring of that cofactor. A pair of these catalytic centers constitutes the catalytic core [PP:PYR]* within these enzymes. Analysis of the structural elements of this catalytic core reveals novel definition of the common amino acid sequences, which are GX@&(G)@XXGQ, and GDGX25-30 within the PP- domain, and the E&(G)@XXG@ within the PYR-domain, where Q, corresponds to a hydrophobic amino acid. This TPP-motif provides a novel tool for annotation of TPP-dependent enzymes useful in advancing functional proteomics.

Molecular cloning, organellar targeting and developmental expression of mitochondrial chaperone HSP60 in Toxoplasma gondii.

PubMed

Toursel, C; Dzierszinski, F; Bernigaud, A; Mortuaire, M; Tomavo, S

2000-12-01

The obligate intracellular protozoan parasite Toxoplasma gondii has a single tubular mitochondrion. During infection, it recruits the host cell's mitochondria abutting to the intracellular vacuole, that contains the parasites. The respective contribution of host and parasitic mitochondria in the intracellular growth of T. gondii remains unknown. Heat shock protein, HSP60 has been reported in all eukaryotes examined, as an essential chaperone required for the folding and multimeric complex assembly of mitochondrial proteins. Here, we report the isolation and molecular characterization of two cDNAs corresponding to a single T. gondii gene coding for HSP60. Using a model fusion protein, preHSP60-chloramphenicol acetyl transferase (CAT), we demonstrate that the classical 22 amino acid mitochondrial presequence and the adjacent 32 amino acids of the mature protein are both required for the in vivo import into T. gondii mitochondria. The T. gondii HSP60 gene composed of five introns and six exons is transcribed into two related but differently spliced transcripts. Whereas the two transcripts can be detected in both developmental stages within the intermediate host, their levels are significantly increased in bradyzoites when compared to tachyzoites. By immunoblot analysis, the predicted 60-kDa protien corresponding to HSP60 was detected in both tachyzoite and bradyzoite forms. Using immunofluorescence assays. the polyclonal antibodies specific to T. gondii HSP60 recognized the mitochondrion in tachyzoites, as expected. In contrast, these antibodies reacted against two unknown vesicular bodies which are distinct from the classical mitochondrial pattern in bradyzoites. Taken together. these expression patterns of mitochondrial chaperone HSP60 suggests stage-specific induction of the respiratory pathway in the protozoan parasite T. gondii.

The microsporidian polar tube: evidence for a third polar tube protein (PTP3) in Encephalitozoon cuniculi.

PubMed

Peuvel, Isabelle; Peyret, Pierre; Méténier, Guy; Vivarès, Christian P; Delbac, Frédéric

2002-06-01

The invasion strategy used by microsporidia is primarily related to spore germination. Small differentiated spores of these fungi-related parasites inject their contents into target cells through the lumen of a rapidly extruded polar tube, as a prerequisite to obligate intracellular development. Previous studies in Encephalitozoon species that infect mammals have identified two major antigenic polar tube proteins (PTP1 and PTP2) which are predicted to contribute to the high tensile strength of the polar tube via an assembly process dependent on disulfide linkages. By immunoscreening of a cDNA library, we found that a novel PTP is encoded by a single transcription unit (3990 bp) located on the chromosome XI of E. cuniculi. PTP3 is predicted to be synthesized as a 1256-amino acid precursor with a cleavable signal peptide. The mature protein lacks cysteine residue and its large acidic core is flanked by highly basic N- and C-terminal regions. Immunolocalization data indicated that PTP3 is involved in the sporoblast-to-spore polar tube biogenesis. A transcriptional up-regulation during sporogony is supported by a strong increase in the relative amount of Ecptp mRNAs within host cells sampled at late post-infection times. To begin to explore polar tube-associated protein interactions, spore proteins were extracted in the presence of SDS and dithiothreitol then incubated with a chemical cross-linker (DSP or sulfo-EGS). A large multimeric complex was formed and shown to contain PTP1, PTP2 and PTP3 with a few other proteins. PTP3 is hypothesized to play a role in the control of the polar tube extrusion as part of a specific response to ionic stimuli.

Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague

PubMed Central

Goodin, Jeremy L.; Nellis, David F.; Powell, Bradford S.; Vyas, Vinay V.; Enama, Jeffrey T.; Wang, Lena C.; Clark, Patrick K.; Giardina, Steven L.; Adamovicz, Jeffery. J.; Michiel, Dennis F.

2009-01-01

The F1-V vaccine antigen, protective against Yersinia pestis, exhibits a strong tendency to multimerize that affects larger-scale manufacture and characterization. In this work, the sole F1-V cysteine was replaced with serine by site-directed mutagenesis for characterization of F1-V non-covalent multimer interactions and protective potency without participation by disulfide-linkages. F1-V and F1-VC424S proteins were over-expressed in Escherichia coli, recovered using mechanical lysis/pH-modulation and purified from urea-solubilized soft inclusion bodies, using successive ion-exchange, ceramic hydroxyapatite, and size-exclusion chromatography. This purification method resulted in up to 2 mg per gram of cell paste of 95% pure, mono-disperse protein having ≤ 0.5 endotoxin units per mg by a kinetic chromogenic limulus amoebocyte lysate reactivity assay. Both F1-V and F1-VC424S were monomeric at pH 10.0 and progressively self-associated as pH conditions decreased to pH 6.0. Solution additives were screened for their ability to inhibit F1-V self-association at pH 6.5. An L-arginine buffer provided the greatest stabilizing effect. Conversion to >500-kDa multimers occurred between pH 6.0 and 5.0. Conditions for efficient F1-V adsorption to the cGMP-compatible Alhydrogel® adjuvant were optimized. Side-by-side evaluation for protective potency against subcutaneous plague infection in mice was conducted for F1-VC424S monomer; cysteine-capped F1-V monomer; cysteine-capped F1-V multimer; and a F1-V standard reported previously. After a two-dose vaccination with 2 × 20 µg of F1-V, respectively, 100, 80, 80, and 70% of injected mice survived a subcutaneous lethal plague challenge with 108 LD50 Y. pestis CO92. Thus, vaccination with F1-V monomer and multimeric forms resulted in significant, and essentially equivalent, protection. PMID:17293124

Expression and characterization of a histidine-rich protein, Hpn: potential for Ni2+ storage in Helicobacter pylori

PubMed Central

Ge, Ruiguang; Watt, Rory M.; Sun, Xuesong; Tanner, Julian A.; He, Qing-Yu; Huang, Jian-Dong; Sun, Hongzhe

2005-01-01

Hpn is a small cytoplasmic protein found in Helicobacter pylori, which binds Ni2+ ions with moderate affinity. Consisting of 60 amino acids, the protein is rich in histidine (28 residues, 46.7%), as well as glutamate, glycine and serine residues (in total 31.7%), and contains short repeating motifs. In the present study, we report the detailed biophysical characterization of the multimeric status and Ni2+-binding properties of purified recombinant Hpn under physiologically relevant conditions. The protein exists as an equilibration of multimeric forms in solution, with 20-mers (approx. 136 kDa) being the predominant species. Using equilibrium dialysis, ICP-MS (inductively coupled plasma MS) and UV/visible spectroscopy, Hpn was found to bind five Ni2+ ions per monomer at pH 7.4, with a dissociation constant (Kd) of 7.1 μM. Importantly, Ni2+ binding to Hpn is reversible: metal is released either in the presence of a chelating ligand such as EDTA, or at a slightly acidic pH (pH for half dissociation, pH1/2 ∼6.3). Ni2+ binding induces conformational changes within the protein, increasing β-sheet and reducing α-helical content, from 22% to 37%, and 20% to 10% respectively. Growth curves of Escherichia coli BL21(DE3) both with and without the hpn gene performed under Ni2+ pressure clearly implied a role for Hpn to protect the cells from higher concentrations of external metal ions. Similarly, the accumulation of Ni2+ in these cells expressing Hpn from a plasmid was approx. 4-fold higher than in uninduced controls or control cultures that lacked the plasmid. Similarly, levels of Ni2+ in wild-type H. pylori 26695 cells were higher than those in H. pylori hpn-deletion mutant strains. Hpn may potentially serve multiple roles inside the bacterium: storage of Ni2+ ions in a ‘reservoir’; donation of Ni2+ to other proteins; and detoxification via sequestration of excess Ni2+. PMID:16164421

The Thiamine-Pyrophosphate-Motif

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Dominiak, Paulina

2004-01-01

Thiamin pyrophosphate (TPP), a derivative of vitamin B1, is a cofactor for enzymes performing catalysis in pathways of energy production including the well known decarboxylation of a-keto acid dehydrogenases followed by transketolation. TPP-dependent enzymes constitute a structurally and functionally diverse group exhibiting multimeric subunit organization, multiple domains and two chemically equivalent catalytic centers. Annotation of functional TPP-dependcnt enzymes, therefore, has not been trivial due to low sequence similarity related to this complex organization. Our approach to analysis of structures of known TPP-dependent enzymes reveals for the first time features common to this group, which we have termed the TPP-motif. The TPP-motif consists of specific spatial arrangements of structural elements and their specific contacts to provide for a flip-flop, or alternate site, enzymatic mechanism of action. Analysis of structural elements entrained in the flip-flop action displayed by TPP-dependent enzymes reveals a novel definition of the common amino acid sequences. These sequences allow for annotation of TPP-dependent enzymes, thus advancing functional proteomics. Further details of three-dimensional structures of TPP-dependent enzymes will be discussed.

The medaka mutation tintachina sheds light on the evolution of V-ATPase B subunits in vertebrates

NASA Astrophysics Data System (ADS)

Müller, Claudia; Maeso, Ignacio; Wittbrodt, Joachim; Martínez-Morales, Juan R.

2013-11-01

Vacuolar-type H+ ATPases (V-ATPases) are multimeric protein complexes that play a universal role in the acidification of intracellular compartments in eukaryotic cells. We have isolated the recessive medaka mutation tintachina (tch), which carries an inactivating modification of the conserved glycine residue (G75R) of the proton pump subunit atp6v1Ba/vatB1. Mutant embryos show penetrant pigmentation defects, massive brain apoptosis and lethality before hatching. Strikingly, an equivalent mutation in atp6v1B1 (G78R) has been reported in a family of patients suffering from distal renal tubular acidosis (dRTA), a hereditary disease that causes metabolic acidosis due to impaired kidney function. This poses the question as to how molecularly identical mutations result in markedly different phenotypes in two vertebrate species. Our work offers an explanation for this phenomenon. We propose that, after successive rounds of whole-genome duplication, the emergence of paralogous copies allowed the divergence of the atp6v1B cis-regulatory control in different vertebrate groups.

Ultrasensitive response motifs: basic amplifiers in molecular signalling networks

PubMed Central

Zhang, Qiang; Bhattacharya, Sudin; Andersen, Melvin E.

2013-01-01

Multi-component signal transduction pathways and gene regulatory circuits underpin integrated cellular responses to perturbations. A recurring set of network motifs serve as the basic building blocks of these molecular signalling networks. This review focuses on ultrasensitive response motifs (URMs) that amplify small percentage changes in the input signal into larger percentage changes in the output response. URMs generally possess a sigmoid input–output relationship that is steeper than the Michaelis–Menten type of response and is often approximated by the Hill function. Six types of URMs can be commonly found in intracellular molecular networks and each has a distinct kinetic mechanism for signal amplification. These URMs are: (i) positive cooperative binding, (ii) homo-multimerization, (iii) multistep signalling, (iv) molecular titration, (v) zero-order covalent modification cycle and (vi) positive feedback. Multiple URMs can be combined to generate highly switch-like responses. Serving as basic signal amplifiers, these URMs are essential for molecular circuits to produce complex nonlinear dynamics, including multistability, robust adaptation and oscillation. These dynamic properties are in turn responsible for higher-level cellular behaviours, such as cell fate determination, homeostasis and biological rhythm. PMID:23615029

Binding Leverage as a Molecular Basis for Allosteric Regulation

PubMed Central

Mitternacht, Simon; Berezovsky, Igor N.

2011-01-01

Allosteric regulation involves conformational transitions or fluctuations between a few closely related states, caused by the binding of effector molecules. We introduce a quantity called binding leverage that measures the ability of a binding site to couple to the intrinsic motions of a protein. We use Monte Carlo simulations to generate potential binding sites and either normal modes or pairs of crystal structures to describe relevant motions. We analyze single catalytic domains and multimeric allosteric enzymes with complex regulation. For the majority of the analyzed proteins, we find that both catalytic and allosteric sites have high binding leverage. Furthermore, our analysis of the catabolite activator protein, which is allosteric without conformational change, shows that its regulation involves other types of motion than those modulated at sites with high binding leverage. Our results point to the importance of incorporating dynamic information when predicting functional sites. Because it is possible to calculate binding leverage from a single crystal structure it can be used for characterizing proteins of unknown function and predicting latent allosteric sites in any protein, with implications for drug design. PMID:21935347

Effects of ACE inhibition and ANG II stimulation on renal Na-Cl cotransporter distribution, phosphorylation, and membrane complex properties

PubMed Central

Lee, Donna H.; Maunsbach, Arvid B.; Riquier-Brison, Anne D.; Nguyen, Mien T. X.; Fenton, Robert A.; Bachmann, Sebastian; Yu, Alan S.

2013-01-01

The renal distal tubule Na-Cl cotransporter (NCC) reabsorbs <10% of the filtered Na+ but is a key control point for blood pressure regulation by angiotensin II (ANG II), angiotensin-converting enzyme inhibitors (ACEI), and thiazide diuretics. This study aimed to determine whether NCC phosphorylation (NCCp) was regulated by acute (20–30 min) treatment with the ACEI captopril (12 μg/min × 20 min) or by a sub-pressor dose of ANG II (20 ng·kg−1·min−1) in Inactin-anesthetized rats. By immuno-EM, NCCp was detected exclusively in or adjacent to apical plama membranes (APM) in controls and after ACEI or ANG II treatment, while NCC total was detected in both APM and subapical cytoplasmic vesicles (SCV) in all conditions. In renal homogenates, neither ACEI nor ANG II treatment altered NCCp abundance, assayed by immunoblot. However, by density gradient fractionation we identified a pool of low-density APM in which NCCp decreased 50% in response to captopril and was restored during ANG II infusion, and another pool of higher-density APM that responded reciprocally, indicative of regulated redistribution between two APM pools. In both pools, NCCp was preferentially localized to Triton-soluble membranes. Blue Native gel electrophoresis established that APM NCCp localized to ∼700 kDa complexes (containing γ-adducin) while unphosphorylated NCC in intracellular membranes primarily localized to ∼400 kDa complexes: there was no evidence for native monomeric or dimeric NCC or NCCp. In summary, this study demonstrates that phosphorylated NCC, localized to multimeric complexes in the APM, redistributes in a regulated manner within the APM in response to ACEI and ANG II. PMID:23114965

Semi-synthesis of a HGF/SF kringle one (K1) domain scaffold generates a potent in vivo MET receptor agonist.

PubMed

Simonneau, Claire; Bérénice Leclercq; Mougel, Alexandra; Adriaenssens, Eric; Paquet, Charlotte; Raibaut, Laurent; Ollivier, Nathalie; Drobecq, Hervé; Marcoux, Julien; Cianférani, Sarah; Tulasne, David; de Jonge, Hugo; Melnyk, Oleg; Vicogne, Jérôme

2015-03-01

The development of MET receptor agonists is an important goal in regenerative medicine, but is limited by the complexity and incomplete understanding of its interaction with HGF/SF (Hepatocyte Growth Factor/Scatter Factor). NK1 is a natural occurring agonist comprising the N-terminal (N) and the first kringle (K1) domains of HGF/SF. In the presence of heparin, NK1 can self-associate into a "head to tail" dimer which is considered as the minimal structural module able to trigger MET dimerization and activation whereas isolated K1 and N domains showed a weak or a complete lack of agonistic activity respectively. Starting from these structural and biological observations, we investigated whether it was possible to recapitulate the biological properties of NK1 using a new molecular architecture of isolated N or K1 domains. Therefore, we engineered multivalent N or K1 scaffolds by combining synthetic and homogeneous site-specifically biotinylated N and K1 domains (NB and K1B) and streptavidin (S). NB alone or in complex failed to activate MET signaling and to trigger cellular phenotypes. Importantly and to the contrary of K1B alone, the semi-synthetic K1B/S complex mimicked NK1 MET agonist activity in cell scattering, morphogenesis and survival phenotypic assays. Impressively, K1B/S complex stimulated in vivo angiogenesis and, when injected in mice, protected the liver against fulminant hepatitis in a MET dependent manner whereas NK1 and HGF were substantially less potent. These data reveal that without N domain, proper multimerization of K1 domain is a promising strategy for the rational design of powerful MET agonists.

COUP-TF (chicken ovalbumin upstream promoter transcription factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding protein.

PubMed Central

Avram, Dorina; Fields, Andrew; Senawong, Thanaset; Topark-Ngarm, Acharawan; Leid, Mark

2002-01-01

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 [CTIP1/Evi9/B cell leukaemia (Bcl) l1a and CTIP2/Bcl11b respectively] are highly related C(2)H(2) zinc finger proteins that are abundantly expressed in brain and the immune system, and are associated with immune system malignancies. A selection procedure was employed to isolate high-affinity DNA binding sites for CTIP1. The core binding site on DNA identified in these studies, 5'-GGCCGG-3' (upper strand), is highly related to the canonical GC box and was bound by a CTIP1 oligomeric complex(es) in vitro. Furthermore, both CTIP1 and CTIP2 repressed transcription of a reporter gene harbouring a multimerized CTIP binding site, and this repression was neither reversed by trichostatin A (an inhibitor of known class I and II histone deacetylases) nor stimulated by co-transfection of a COUP-TF family member. These results demonstrate that CTIP1 is a sequence-specific DNA binding protein and a bona fide transcriptional repressor that is capable of functioning independently of COUP-TF family members. These findings may be relevant to the physiological and/or pathological action(s) of CTIPs in cells that do not express COUP-TF family members, such as cells of the haematopoietic and immune systems. PMID:12196208

Genetic variations in the DNA replication origins of human papillomavirus family correlate with their oncogenic potential.

PubMed

Yilmaz, Gulden; Biswas-Fiss, Esther E; Biswas, Subhasis B

2018-04-01

Human papillomaviruses (HPVs) encompass a large family of viruses that range from benign to highly carcinogenic. The crucial differences between benign and carcinogenic types of HPV remain unknown, except that the two HPV types differ in the frequency of DNA replication. We have systematically analyzed the mechanism of HPV DNA replication initiation in low-risk and high-risk HPVs. Our results demonstrate that HPV-encoded E2 initiator protein and its four binding sites in the replication origin play pivotal roles in determining the destiny of the HPV-infected cell. We have identified strain-specific single nucleotide variations in E2 binding sites found only in the high-risk HPVs. We have demonstrated that these variations result in attenuated formation of the E2-DNA complex. E2 binding to these sites is linked to the activation of the DNA replication origin as well as initiation of DNA replication. Both electrophoretic mobility shift assay and atomic force microscopy studies demonstrated that binding of E2 from either low- or high-risk HPVs with variant binding sequences lacked multimeric E2-DNA complex formation in vitro. These results provided a molecular basis of differential DNA replication in the two types of HPVs and pointed to a correlation with the development of cancer. Copyright © 2017. Published by Elsevier B.V.

The Molecular Chaperone HSPA2 Plays a Key Role in Regulating the Expression of Sperm Surface Receptors That Mediate Sperm-Egg Recognition

PubMed Central

Redgrove, Kate A.; Nixon, Brett; Baker, Mark A.; Hetherington, Louise; Baker, Gordon; Liu, De-Yi; Aitken, R. John

2012-01-01

A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm–egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm–zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility. PMID:23209833

Der p 5 Crystal Structure Provides Insight into the Group 5 Dust Mite Allergens

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

Mueller, G.; Gosavi, R; Krahn, J

2010-01-01

Group 5 allergens from house dust mites elicit strong IgE antibody binding in mite-allergic patients. The structure of Der p 5 was determined by x-ray crystallography to better understand the IgE epitopes, to investigate the biologic function in mites, and to compare with the conflicting published Blo t 5 structures, designated 2JMH and 2JRK in the Protein Data Bank. Der p 5 is a three-helical bundle similar to Blo t 5, but the interactions of the helices are more similar to 2JMH than 2JRK. The crystallographic asymmetric unit contains three dimers of Der p 5 that are not exactly alike.more » Solution scattering techniques were used to assess the multimeric state of Der p 5 in vitro and showed that the predominant state was monomeric, similar to Blo t 5, but larger multimeric species are also present. In the crystal, the formation of the Der p 5 dimer creates a large hydrophobic cavity of {approx}3000 {angstrom}{sup 3} that could be a ligand-binding site. Many allergens are known to bind hydrophobic ligands, which are thought to stimulate the innate immune system and have adjuvant-like effects on IgE-mediated inflammatory responses.« less

Bioluminescence Resonance Energy Transfer System for Measuring Dynamic Protein-Protein Interactions in Bacteria

PubMed Central

Cui, Boyu; Wang, Yao; Song, Yunhong; Wang, Tietao; Li, Changfu; Wei, Yahong

2014-01-01

ABSTRACT Protein-protein interactions are important for virtually every biological process, and a number of elegant approaches have been designed to detect and evaluate such interactions. However, few of these methods allow the detection of dynamic and real-time protein-protein interactions in bacteria. Here we describe a bioluminescence resonance energy transfer (BRET) system based on the bacterial luciferase LuxAB. We found that enhanced yellow fluorescent protein (eYFP) accepts the emission from LuxAB and emits yellow fluorescence. Importantly, BRET occurred when LuxAB and eYFP were fused, respectively, to the interacting protein pair FlgM and FliA. Furthermore, we observed sirolimus (i.e., rapamycin)-inducible interactions between FRB and FKBP12 and a dose-dependent abolishment of such interactions by FK506, the ligand of FKBP12. Using this system, we showed that osmotic stress or low pH efficiently induced multimerization of the regulatory protein OmpR and that the multimerization induced by low pH can be reversed by a neutralizing agent, further indicating the usefulness of this system in the measurement of dynamic interactions. This method can be adapted to analyze dynamic protein-protein interactions and the importance of such interactions in bacterial processes such as development and pathogenicity. PMID:24846380

Thermal, Chemical and pH Induced Denaturation of a Multimeric β-Galactosidase Reveals Multiple Unfolding Pathways

PubMed Central

Kishore, Devesh; Kundu, Suman; Kayastha, Arvind M.

2012-01-01

Background In this case study, we analysed the properties of unfolded states and pathways leading to complete denaturation of a multimeric chick pea β-galactosidase (CpGAL), as obtained from treatment with guanidium hydrochloride, urea, elevated temperature and extreme pH. Methodology/Principal Findings CpGAL, a heterodimeric protein with native molecular mass of 85 kDa, belongs to α+β class of protein. The conformational stability and thermodynamic parameters of CpGAL unfolding in different states were estimated and interpreted using circular dichroism and fluorescence spectroscopic measurements. The enzyme was found to be structurally and functionally stable in the entire pH range and upto 50°C temperature. Further increase in temperature induces unfolding followed by aggregation. Chemical induced denaturation was found to be cooperative and transitions were irreversible, non-coincidental and sigmoidal. Free energy of protein unfolding (ΔG0) and unfolding constant (Kobs) were also calculated for chemically denatured CpGAL. Significance The protein seems to use different pathways for unfolding in different environments and is a classical example of how the environment dictates the path a protein might take to fold while its amino acid sequence only defines its final three-dimensional conformation. The knowledge accumulated could be of immense biotechnological significance as well. PMID:23185611

WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity.

PubMed

Li, Qiuhong; Chang, Leifu; Aibara, Shintaro; Yang, Jing; Zhang, Ziguo; Barford, David

2016-09-20

The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin-RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for destruction via the ubiquitin proteasome system. The APC/C assembly comprises two scaffolding subcomplexes: the platform and the TPR lobe that together coordinate the juxtaposition of the catalytic and substrate-recognition modules. The platform comprises APC/C subunits Apc1, Apc4, Apc5, and Apc15. Although the role of Apc1 as an APC/C scaffolding subunit has been characterized, its specific functions in contributing toward APC/C catalytic activity are not fully understood. Here, we report the crystal structure of the N-terminal domain of human Apc1 (Apc1N) determined at 2.2-Å resolution and provide an atomic-resolution description of the architecture of its WD40 (WD40 repeat) domain (Apc1(WD40)). To understand how Apc1(WD40) contributes to APC/C activity, a mutant form of the APC/C with Apc1(WD40) deleted was generated and evaluated biochemically and structurally. We found that the deletion of Apc1(WD40) abolished the UbcH10-dependent ubiquitination of APC/C substrates without impairing the Ube2S-dependent ubiquitin chain elongation activity. A cryo-EM structure of an APC/C-Cdh1 complex with Apc1(WD40) deleted showed that the mutant APC/C is locked into an inactive conformation in which the UbcH10-binding site of the catalytic module is inaccessible. Additionally, an EM density for Apc15 is not visible. Our data show that Apc1(WD40) is required to mediate the coactivator-induced conformational change of the APC/C that is responsible for stimulating APC/C catalytic activity by promoting UbcH10 binding. In contrast, Ube2S activity toward APC/C substrates is not dependent on the initiation-competent conformation of the APC/C.

WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity

PubMed Central

Li, Qiuhong; Chang, Leifu; Aibara, Shintaro; Yang, Jing; Zhang, Ziguo; Barford, David

2016-01-01

The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin–RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for destruction via the ubiquitin proteasome system. The APC/C assembly comprises two scaffolding subcomplexes: the platform and the TPR lobe that together coordinate the juxtaposition of the catalytic and substrate-recognition modules. The platform comprises APC/C subunits Apc1, Apc4, Apc5, and Apc15. Although the role of Apc1 as an APC/C scaffolding subunit has been characterized, its specific functions in contributing toward APC/C catalytic activity are not fully understood. Here, we report the crystal structure of the N-terminal domain of human Apc1 (Apc1N) determined at 2.2-Å resolution and provide an atomic-resolution description of the architecture of its WD40 (WD40 repeat) domain (Apc1WD40). To understand how Apc1WD40 contributes to APC/C activity, a mutant form of the APC/C with Apc1WD40 deleted was generated and evaluated biochemically and structurally. We found that the deletion of Apc1WD40 abolished the UbcH10-dependent ubiquitination of APC/C substrates without impairing the Ube2S-dependent ubiquitin chain elongation activity. A cryo-EM structure of an APC/C–Cdh1 complex with Apc1WD40 deleted showed that the mutant APC/C is locked into an inactive conformation in which the UbcH10-binding site of the catalytic module is inaccessible. Additionally, an EM density for Apc15 is not visible. Our data show that Apc1WD40 is required to mediate the coactivator-induced conformational change of the APC/C that is responsible for stimulating APC/C catalytic activity by promoting UbcH10 binding. In contrast, Ube2S activity toward APC/C substrates is not dependent on the initiation-competent conformation of the APC/C. PMID:27601667

Fission yeast translation initiation factor 3 subunit eIF3h is not essential for global translation initiation, but deletion of eif3h+ affects spore formation.

PubMed

Ray, Anirban; Bandyopadhyay, Amitabha; Matsumoto, Tomohiro; Deng, Haiteng; Maitra, Umadas

2008-11-01

The fission yeast Schizosaccharomyces pombe homologue of the p40/eIF3h subunit of mammalian translation initiation factor eIF3 has been characterized in this study. We show that this protein physically associates with the 40S ribosomal particles as a constituent of the multimeric eIF3 protein complex, which consists of all five known eIF3 core subunits (eIF3a, eIF3b, eIF3c, eIF3g and eIF3i) as well as the five non-core subunits (eIF3d, eIF3e, eIF3f, eIF3h and eIF3m) that constitute an eIF3 holocomplex in fission yeast. However, affinity purification of eIF3 from fission yeast cells expressing TAP-tagged eIF3h suggests the presence of distinct forms of eIF3 that differ in their composition of the non-core subunits. Further characterization of eIF3h shows that strains lacking eif3h(+) (eif3hDelta) are viable and show no gross defects, either in vegetative growth or in the rate of in vivo protein synthesis. Polysome profile analysis shows no apparent defects in translation initiation. Furthermore, deletion of eif3h(+) does not affect the ability of the other eIF3 subunits to remain associated with one another in a tight protein complex similar to the situation in wild-type cells. Additionally, we show that human eIF3h can functionally substitute fission yeast eIF3h in complementing in vivo a genetic deletion of eif3h(+). Interestingly, mutant eif3hDelta cells show several prominent phenotypic properties. They are hypersensitive to caffeine and highly defective in meiosis, producing either no spores or incomplete tetrads with a very high frequency. The implications of these results in relation to the functions of eIF3h in Sz. pombe are discussed. (c) 2008 John Wiley & Sons, Ltd.

Use of the interior cavity of the P22 capsid for site-specific initiation of atom-transfer radical polymerization with high-density cargo loading

NASA Astrophysics Data System (ADS)

Lucon, Janice; Qazi, Shefah; Uchida, Masaki; Bedwell, Gregory J.; Lafrance, Ben; Prevelige, Peter E.; Douglas, Trevor

2012-10-01

Virus-like particles (VLPs) have emerged as important and versatile architectures for chemical manipulation in the development of functional hybrid nanostructures. Here we demonstrate a successful site-selective initiation of atom-transfer radical polymerization reactions to form an addressable polymer constrained within the interior cavity of a VLP. Potentially, this protein-polymer hybrid of P22 and cross-linked poly(2-aminoethyl methacrylate) could be useful as a new high-density delivery vehicle for the encapsulation and delivery of small-molecule cargos. In particular, the encapsulated polymer can act as a scaffold for the attachment of small functional molecules, such as fluorescein dye or the magnetic resonance imaging (MRI) contrast agent Gd-diethylenetriaminepentacetate, through reactions with its pendant primary amine groups. Using this approach, a significant increase in the labelling density of the VLP, compared to that of previous modifications of VLPs, can be achieved. These results highlight the use of multimeric protein-polymer conjugates for their potential utility in the development of VLP-based MRI contrast agents with the possibility of loading other cargos.

A map of human PRDM9 binding provides evidence for novel behaviors of PRDM9 and other zinc-finger proteins in meiosis

PubMed Central

Noor, Nudrat; Bitoun, Emmanuelle; Tumian, Afidalina; Imbeault, Michael; Chapman, J Ross; Aricescu, A Radu

2017-01-01

PRDM9 binding localizes almost all meiotic recombination sites in humans and mice. However, most PRDM9-bound loci do not become recombination hotspots. To explore factors that affect binding and subsequent recombination outcomes, we mapped human PRDM9 binding sites in a transfected human cell line and measured PRDM9-induced histone modifications. These data reveal varied DNA-binding modalities of PRDM9. We also find that human PRDM9 frequently binds promoters, despite their low recombination rates, and it can activate expression of a small number of genes including CTCFL and VCX. Furthermore, we identify specific sequence motifs that predict consistent, localized meiotic recombination suppression around a subset of PRDM9 binding sites. These motifs strongly associate with KRAB-ZNF protein binding, TRIM28 recruitment, and specific histone modifications. Finally, we demonstrate that, in addition to binding DNA, PRDM9's zinc fingers also mediate its multimerization, and we show that a pair of highly diverged alleles preferentially form homo-multimers. PMID:29072575

Novel adapter proteins that link the human GM-CSF receptor to the phosphatidylino-sitol 3-kinase and Shc/Grb2/ras signaling pathways.

PubMed

Jücker, M; Feldman, R A

1996-01-01

We have used a human GM-CSF-dependent hematopoietic cell line that responds to physiological concentrations of hGM-CSF to analyze a set of signaling events that occur in normal myelopoiesis and whose deregulation may lead to leukemogenesis. Stimulation of these cells with hGM-CSF induced the assembly of multimeric complexes that contained known and novel phosphotyrosyl proteins. One of the new proteins was a major phosphotyrosyl substrate of 76-85 kDa (p80) that was directly associated with the p85 subunit of phosphatidylinositol (PI) 3-kinase through the SH2 domains of p85. p80 also associated with the beta subunit of the activated hGM-CSF receptor, and assembly of this complex correlated with activation of PI 3-kinase. A second phosphotyrosyl protein we identified, p140, associated with the Shc and Grb2 adapter proteins by direct binding to a novel phosphotyrosine-interacting domain located at the N-terminus of Shc. and to the SH3 domains of Grb2, respectively. The Shc/p140/Grb2 complex was found to be constitutively activated in acute myeloid leukemia cells, indicating that activation of this pathway may be a necessary step in the development of some leukemias. The p80/p85/PI 3-kinase and the Shc/Grb2/p140 complexes were tightly associated with Src family kinases, which were prime candidates for phosphorylation of Shc, p80, p140 and other phosphotyrosyl substrates present in these complexes. Our studies suggest that p80 and p140 may link the hGM-CSF receptor to the PI 3-kinase and Shc/Grb2/ras signaling pathways, respectively, and that abnormal activation of hGM-CSF-dependent targets may play a role in leukemogenesis.

From an old remedy to a magic bullet: molecular mechanisms underlying the therapeutic effects of arsenic in fighting leukemia

PubMed Central

Zhou, Guang-Biao; Zhang, Xiao-Wei; Mao, Jian-Hua; de Thé, Hugues

2011-01-01

Arsenic had been used in treating malignancies from the 18th to mid-20th century. In the past 3 decades, arsenic was revived and shown to be able to induce complete remission and to achieve, when combined with all-trans retinoic acid and chemotherapy, a 5-year overall survival of 90% in patients with acute promyelocytic leukemia driven by the t(15;17) translocation-generated promyelocytic leukemia–retinoic acid receptor α (PML-RARα) fusion. Molecularly, arsenic binds thiol residues and induces the formation of reactive oxygen species, thus affecting numerous signaling pathways. Interestingly, arsenic directly binds the C3HC4 zinc finger motif in the RBCC domain of PML and PML-RARα, induces their homodimerization and multimerization, and enhances their interaction with the SUMO E2 conjugase Ubc9, facilitating subsequent sumoylation/ubiquitination and proteasomal degradation. Arsenic-caused intermolecular disulfide formation in PML also contributes to PML-multimerization. All-trans retinoic acid, which targets PML-RARα for degradation through its RARα moiety, synergizes with arsenic in eliminating leukemia-initiating cells. Arsenic perturbs a number of proteins involved in other hematologic malignancies, including chronic myeloid leukemia and adult T-cell leukemia/lymphoma, whereby it may bring new therapeutic benefits. The successful revival of arsenic in acute promyelocytic leukemia, together with modern mechanistic studies, has thus allowed a new paradigm to emerge in translational medicine. PMID:21422471

Preservation of viral genomes in 700-y-old caribou feces from a subarctic ice patch

PubMed Central

Chen, Li-Fang; Zhou, Yanchen; Shapiro, Beth; Stiller, Mathias; Varsani, Arvind; Kondov, Nikola O.; Wong, Walt; Deng, Xutao; Andrews, Thomas D.; Moorman, Brian J.; Meulendyk, Thomas; MacKay, Glen; Gilbertson, Robert L.; Delwart, Eric

2014-01-01

Viruses preserved in ancient materials provide snapshots of past viral diversity and a means to trace viral evolution through time. Here, we use a metagenomics approach to identify filterable and nuclease-resistant nucleic acids preserved in 700-y-old caribou feces frozen in a permanent ice patch. We were able to recover and characterize two viruses in replicated experiments performed in two different laboratories: a small circular DNA viral genome (ancient caribou feces associated virus, or aCFV) and a partial RNA viral genome (Ancient Northwest Territories cripavirus, or aNCV). Phylogenetic analysis identifies aCFV as distantly related to the plant-infecting geminiviruses and the fungi-infecting Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 and aNCV as within the insect-infecting Cripavirus genus. We hypothesize that these viruses originate from plant material ingested by caribou or from flying insects and that their preservation can be attributed to protection within viral capsids maintained at cold temperatures. To investigate the tropism of aCFV, we used the geminiviral reverse genetic system and introduced a multimeric clone into the laboratory model plant Nicotiana benthamiana. Evidence for infectivity came from the detection of viral DNA in newly emerged leaves and the precise excision of the viral genome from the multimeric clones in inoculated leaves. Our findings indicate that viral genomes may in some circumstances be protected from degradation for centuries. PMID:25349412

Immature HIV-1 lattice assembly dynamics are regulated by scaffolding from nucleic acid and the plasma membrane

PubMed Central

Pak, Alexander J.; Grime, John M. A.; Sengupta, Prabuddha; Chen, Antony K.; Durumeric, Aleksander E. P.; Srivastava, Anand; Yeager, Mark; Briggs, John A. G.; Lippincott-Schwartz, Jennifer; Voth, Gregory A.

2017-01-01

The packaging and budding of Gag polyprotein and viral RNA is a critical step in the HIV-1 life cycle. High-resolution structures of the Gag polyprotein have revealed that the capsid (CA) and spacer peptide 1 (SP1) domains contain important interfaces for Gag self-assembly. However, the molecular details of the multimerization process, especially in the presence of RNA and the cell membrane, have remained unclear. In this work, we investigate the mechanisms that work in concert between the polyproteins, RNA, and membrane to promote immature lattice growth. We develop a coarse-grained (CG) computational model that is derived from subnanometer resolution structural data. Our simulations recapitulate contiguous and hexameric lattice assembly driven only by weak anisotropic attractions at the helical CA–SP1 junction. Importantly, analysis from CG and single-particle tracking photoactivated localization (spt-PALM) trajectories indicates that viral RNA and the membrane are critical constituents that actively promote Gag multimerization through scaffolding, while overexpression of short competitor RNA can suppress assembly. We also find that the CA amino-terminal domain imparts intrinsic curvature to the Gag lattice. As a consequence, immature lattice growth appears to be coupled to the dynamics of spontaneous membrane deformation. Our findings elucidate a simple network of interactions that regulate the early stages of HIV-1 assembly and budding. PMID:29114055

A Protein Linkage Map of the P2 Nonstructural Proteins of Poliovirus

PubMed Central

Cuconati, Andrea; Xiang, Wenkai; Lahser, Frederick; Pfister, Thomas; Wimmer, Eckard

1998-01-01

The yeast two-hybrid system was used to catalog all detectable interactions among the P2 nonstructural cleavage products of poliovirus type 1 (Mahoney). Evidence has been obtained for specific associations among 2Apro, 2BC, 2C, and 2B. Specifically, 2Apro can interact with itself and 2BC and its cleavage products (2B and 2C) interact in all possible combinations, with the exception of 2C/2C. Detected interactions were confirmed in vitro by a glutathione S-transferase pulldown assay, which allowed us to detect 2C/2C association. trans-dominant-negative mutants of 2B (K. Johnson and P. J. Sarnow, J. Virol. 65:4341–4349, 1991) were examined and were found to retain interaction with wild-type 2B, perhaps reflecting a need for 2B multimerization in viral RNA replication. The multimerization of 2B was examined further by screening a mutagenized library for 2B variants that have lost the ability to bind wild-type 2B. The screen identified two nonconservative missense mutations within a central hydrophobic region, as well as truncations and frameshifts that implicate the C terminus in homointeraction. Introduction of the missense mutations into the genome of the virus conferred a quasi-infectious phenotype, an observation strongly suggesting that the 2B/2B interaction is required for replication of the viral genome. PMID:9445030

Antibody engineering of a cytotoxic monoclonal antibody 84 against human embryonic stem cells: Investigating the effects of multivalency on cytotoxicity.

PubMed

Klement, Maximilian; Zheng, Jiyun; Liu, Chengcheng; Tan, Heng-Liang; Wong, Victor Vai Tak; Choo, Andre Boon-Hwa; Lee, Dong-Yup; Ow, Dave Siak-Wei

2017-02-10

Antibody fragments have shown targeted specificity to their antigens, but only modest tissue retention times in vivo and in vitro. Multimerization has been used as a protein engineering tool to increase the number of binding units and thereby enhance the efficacy and retention time of antibody fragments. In this work, we explored the effects of valency using a series of self-assembling polypeptides based on the GCN4 leucine zipper multimerization domain fused to a single-chain variable fragment via an antibody upper hinge sequence. Four engineered antibody fragments with a valency from one to four antigen-binding units of a cytotoxic monoclonal antibody 84 against human embryonic stem cells (hESC) were constructed. We hypothesized that higher cytotoxicity would be observed for fragments with increased valency. Flow cytometry analysis revealed that the trimeric and tetrameric engineered antibody fragments resulted in the highest degree of cytotoxicity to the undifferentiated hESC, while the engineered antibody fragments were observed to have improved tissue penetration into cell clusters. Thus, a trade off was made for the trimeric versus tetrameric fragment due to improved tissue penetration. These results have direct implications for antibody-mediated removal of undifferentiated hESC during regenerative medicine and cell therapy. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

Characteristic of entire corneal topography and tomography for the detection of sub-clinical keratoconus with Zernike polynomials using Pentacam.

PubMed

Xu, Zhe; Li, Weibo; Jiang, Jun; Zhuang, Xiran; Chen, Wei; Peng, Mei; Wang, Jianhua; Lu, Fan; Shen, Meixiao; Wang, Yuanyuan

2017-11-28

The study aimed to characterize the entire corneal topography and tomography for the detection of sub-clinical keratoconus (KC) with a Zernike application method. Normal subjects (n = 147; 147 eyes), sub-clinical KC patients (n = 77; 77 eyes), and KC patients (n = 139; 139 eyes) were imaged with the Pentacam HR system. The entire corneal data of pachymetry and elevation of both the anterior and posterior surfaces were exported from the Pentacam HR software. Zernike polynomials fitting was used to quantify the 3D distribution of the corneal thickness and surface elevation. The root mean square (RMS) values for each order and the total high-order irregularity were calculated. Multimeric discriminant functions combined with individual indices were built using linear step discriminant analysis. Receiver operating characteristic curves determined the diagnostic accuracy (area under the curve, AUC). The 3rd-order RMS of the posterior surface (AUC: 0.928) obtained the highest discriminating capability in sub-clinical KC eyes. The multimeric function, which consisted of the Zernike fitting indices of corneal posterior elevation, showed the highest discriminant ability (AUC: 0.951). Indices generated from the elevation of posterior surface and thickness measurements over the entire cornea using the Zernike method based on the Pentacam HR system were able to identify very early KC.

The Recombinant Inhibitor of DNA Binding Id2 Forms Multimeric Structures via the Helix-Loop-Helix Domain and the Nuclear Export Signal.

PubMed

Roschger, Cornelia; Schubert, Mario; Regl, Christof; Andosch, Ancuela; Marquez, Augusto; Berger, Thomas; Huber, Christian G; Lütz-Meindl, Ursula; Cabrele, Chiara

2018-04-07

The inhibitor of DNA binding and cell differentiation 2 (Id2) is a helix-loop-helix (HLH) protein that acts as negative dominant regulator of basic-HLH transcription factors during development and in cancer. The structural properties of Id2 have been investigated so far by using synthetic or recombinant fragments reproducing single domains (N-terminus, HLH, C-terminus): the HLH domain tends to dimerize into a four-helix bundle, whereas the flanking regions are flexible. In this work, the intact protein was expressed in E. coli , solubilized from inclusion bodies with urea, purified and dissolved in water at pH~4. Under these conditions, Id2 was obtained with both cysteine residues disulfide-bonded to β-mercaptoethanol that was present during the solubilization process. Moreover, it existed in a self-assembled state, in which the N-terminus remained highly flexible, while the HLH domain and, surprisingly, part of the C-terminus, which corresponds to the nuclear export signal (NES), both were involved in slowly tumbling, rigid structures. The protein oligomers also formed twisted fibrils that were several micrometers long and up to 80 nm thick. These results show that self-assembly decreases the backbone flexibility of those two protein regions (HLH and NES) that are important for interaction with basic-HLH transcription factors or for nucleocytoplasmic shuttling.

Structural assemblies of the di- and oligomeric G-protein coupled receptor TGR5 in live cells: an MFIS-FRET and integrative modelling study

NASA Astrophysics Data System (ADS)

Greife, Annemarie; Felekyan, Suren; Ma, Qijun; Gertzen, Christoph G. W.; Spomer, Lina; Dimura, Mykola; Peulen, Thomas O.; Wöhler, Christina; Häussinger, Dieter; Gohlke, Holger; Keitel, Verena; Seidel, Claus A. M.

2016-11-01

TGR5 is the first identified bile acid-sensing G-protein coupled receptor, which has emerged as a potential therapeutic target for metabolic disorders. So far, structural and multimerization properties are largely unknown for TGR5. We used a combined strategy applying cellular biology, Multiparameter Image Fluorescence Spectroscopy (MFIS) for quantitative FRET analysis, and integrative modelling to obtain structural information about dimerization and higher-order oligomerization assemblies of TGR5 wildtype (wt) and Y111 variants fused to fluorescent proteins. Residue 111 is located in transmembrane helix 3 within the highly conserved ERY motif. Co-immunoprecipitation and MFIS-FRET measurements with gradually increasing acceptor to donor concentrations showed that TGR5 wt forms higher-order oligomers, a process disrupted in TGR5 Y111A variants. From the concentration dependence of the MFIS-FRET data we conclude that higher-order oligomers - likely with a tetramer organization - are formed from dimers, the smallest unit suggested for TGR5 Y111A variants. Higher-order oligomers likely have a linear arrangement with interaction sites involving transmembrane helix 1 and helix 8 as well as transmembrane helix 5. The latter interaction is suggested to be disrupted by the Y111A mutation. The proposed model of TGR5 oligomer assembly broadens our view of possible oligomer patterns and affinities of class A GPCRs.

Identification of a tetramerization domain in the C terminus of the vanilloid receptor.

PubMed

García-Sanz, Nuria; Fernández-Carvajal, Asia; Morenilla-Palao, Cruz; Planells-Cases, Rosa; Fajardo-Sánchez, Emmanuel; Fernández-Ballester, Gregorio; Ferrer-Montiel, Antonio

2004-06-09

TRPV1 (transient receptor potential vanilloid receptor subtype 1) is a member of the TRP channel family gated by vanilloids, protons, and heat. Structurally, TRPV1 appears to be a tetramer formed by the assembly of four identical subunits around a central aqueous pore. The molecular determinants that govern its subunit oligomerization remain elusive. Here, we report the identification of a segment comprising 684Glu-721Arg (referred to as the TRP-like domain) in the C terminus of TRPV1 as an association domain (AD) of the protein. Purified recombinant C terminus of TRPV1 (TRPV1-C) formed discrete and stable multimers in vitro. Yeast two-hybrid and pull-down assays showed that self-association of the TRPV1-C is blocked when segment 684Glu-721Arg is deleted. Biochemical and immunological analysis indicate that removal of the AD from full-length TRPV1 monomers blocks the formation of stable heteromeric assemblies with wild-type TRPV1 subunits. Deletion of the AD in a poreless TRPV1 subunit suppressed its robust dominant-negative phenotype. Together, these findings are consistent with the tenet that the TRP-like domain in TRPV1 is a molecular determinant of the tetramerization of receptor subunits into functional channels. Our observations suggest that the homologous TRP domain in the TRP protein family may function as a general, evolutionary conserved AD involved in subunit multimerization.

Controllable molecular motors engineered from myosin and RNA

NASA Astrophysics Data System (ADS)

Omabegho, Tosan; Gurel, Pinar S.; Cheng, Clarence Y.; Kim, Laura Y.; Ruijgrok, Paul V.; Das, Rhiju; Alushin, Gregory M.; Bryant, Zev

2018-01-01

Engineering biomolecular motors can provide direct tests of structure-function relationships and customized components for controlling molecular transport in artificial systems1 or in living cells2. Previously, synthetic nucleic acid motors3-5 and modified natural protein motors6-10 have been developed in separate complementary strategies to achieve tunable and controllable motor function. Integrating protein and nucleic-acid components to form engineered nucleoprotein motors may enable additional sophisticated functionalities. However, this potential has only begun to be explored in pioneering work harnessing DNA scaffolds to dictate the spacing, number and composition of tethered protein motors11-15. Here, we describe myosin motors that incorporate RNA lever arms, forming hybrid assemblies in which conformational changes in the protein motor domain are amplified and redirected by nucleic acid structures. The RNA lever arm geometry determines the speed and direction of motor transport and can be dynamically controlled using programmed transitions in the lever arm structure7,9. We have characterized the hybrid motors using in vitro motility assays, single-molecule tracking, cryo-electron microscopy and structural probing16. Our designs include nucleoprotein motors that reversibly change direction in response to oligonucleotides that drive strand-displacement17 reactions. In multimeric assemblies, the controllable motors walk processively along actin filaments at speeds of 10-20 nm s-1. Finally, to illustrate the potential for multiplexed addressable control, we demonstrate sequence-specific responses of RNA variants to oligonucleotide signals.

Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes.

PubMed

Xxxx, Patmawati; Minamihata, Kosuke; Tatsuke, Tsuneyuki; Lee, Jae Man; Kusakabe, Takahiro; Kamiya, Noriho

2018-06-01

Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The C-terminal region of the non-structural protein 2B from Hepatitis A Virus demonstrates lipid-specific viroporin-like activity

NASA Astrophysics Data System (ADS)

Shukla, Ashutosh; Dey, Debajit; Banerjee, Kamalika; Nain, Anshu; Banerjee, Manidipa

2015-10-01

Viroporins are virally encoded, membrane-active proteins, which enhance viral replication and assist in egress of viruses from host cells. The 2B proteins in the picornaviridae family are known to have viroporin-like properties, and play critical roles during virus replication. The 2B protein of Hepatitis A Virus (2B), an unusual picornavirus, is somewhat dissimilar from its analogues in several respects. HAV 2B is approximately 2.5 times the length of other 2B proteins, and does not disrupt calcium homeostasis or glycoprotein trafficking. Additionally, its membrane penetrating properties are not yet clearly established. Here we show that the membrane interacting activity of HAV 2B is localized in its C-terminal region, which contains an alpha-helical hairpin motif. We show that this region is capable of forming small pores in membranes and demonstrates lipid specific activity, which partially rationalizes the intracellular localization of full-length 2B. Using a combination of biochemical assays and molecular dynamics simulation studies, we also show that HAV 2B demonstrates a marked propensity to dimerize in a crowded environment, and probably interacts with membranes in a multimeric form, a hallmark of other picornavirus viroporins. In sum, our study clearly establishes HAV 2B as a bona fide viroporin in the picornaviridae family.

Iron-sulfur cluster biogenesis in mammalian cells: new insights into the molecular mechanisms of cluster delivery

PubMed Central

Maio, Nunziata; Rouault, Tracey. A.

2014-01-01

Iron-sulfur (Fe-S) clusters are ancient, ubiquitous cofactors composed of iron and inorganic sulfur. The combination of the chemical reactivity of iron and sulfur, together with many variations of cluster composition, oxidation states and protein environments, enables Fe-S clusters to participate in numerous biological processes. Fe-S clusters are essential to redox catalysis in nitrogen fixation, mitochondrial respiration and photosynthesis, to regulatory sensing in key metabolic pathways (i. e. cellular iron homeostasis and oxidative stress response), and to the replication and maintenance of the nuclear genome. Fe-S cluster biogenesis is a multistep process that involves a complex sequence of catalyzed protein- protein interactions and coupled conformational changes between the components of several dedicated multimeric complexes. Intensive studies of the assembly process have clarified key points in the biogenesis of Fe-S proteins. However several critical questions still remain, such as: what is the role of frataxin? Why do some defects of Fe-S cluster biogenesis cause mitochondrial iron overload? How are specific Fe-S recipient proteins recognized in the process of Fe-S transfer? This review focuses on the basic steps of Fe-S cluster biogenesis, drawing attention to recent advances achieved on the identification of molecular features that guide selection of specific subsets of nascent Fe-S recipients by the cochaperone HSC20. Additionally, it outlines the distinctive phenotypes of human diseases due to mutations in the components of the basic pathway. PMID:25245479

Characterization of Protein Detergent Complexes by NMR, Light Scattering, and Analytical Ultracentrifugation

PubMed Central

Maslennikov, Innokentiy; Krupa, Martin; Dickson, Christopher; Esquivies, Luis; Blain, Katherine; Kefala, Georgia; Choe, Senyon; Kwiatkowski, Witek

2009-01-01

Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent complexes (PDCs) (Maslennikov et al., 2007), we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably, this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore more effective than centrifugal ultrafiltration devices. PMID:19214777

The gene for replication factor C subunit 2 (RFC2) is within the 7q11.23 Williams syndrome deletion

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

Peoples, R.; Perez-Jurado, L.; Francke, U.

1996-06-01

Williams syndrome (WS) is a developmental disorder with multiple system manifestations, including supraval var aortic stenosis (SVAS), peripheral pulmonic stenosis, connective tissue abnormalities, short stature, characteristic personality profile and cognitive deficits, and variable hypercalcemia in infancy. It is caused by heterozygosity for a chromosomal deletion of part of band 7q11.23 including the elastin locus (ELN). Since disruption of the ELN gene causes autosomal dominant SVAS, it is assumed that ELN haploinsufficiency is responsible for the cardiovascular features of WS. The deletion that extends from the ELN locus in both directions is {ge}200 kb in size, although estimates of {ge}2 Mbmore » are suggested by high-resolution chromosome banding and physical mapping studies. We have searched for additional dosage-sensitive genes within the deletion that may be responsible for the noncardiovascular features. We report here that the gene for replication factor C subunit 2 (RFC2) maps within the WS deletion region and was found to be deleted in all of 18 WS patients studied. The protein product of RFC2 is part of a multimeric complex involved in DNA elongation during replication. 14 refs., 3 figs.« less

The Adenovirus E1A C Terminus Suppresses a Delayed Antiviral Response and Modulates RAS Signaling.

PubMed

Zemke, Nathan R; Berk, Arnold J

2017-12-13

The N-terminal half of adenovirus e1a assembles multimeric complexes with host proteins that repress innate immune responses and force host cells into S-phase. In contrast, the functions of e1a's C-terminal interactions with FOXK, DCAF7, and CtBP are unknown. We found that these interactions modulate RAS signaling, and that a single e1a molecule must bind all three of these host proteins to suppress activation of a subset of IFN-stimulated genes (ISGs). These ISGs were otherwise induced in primary respiratory epithelial cells at 12 hr p.i. This delayed activation of ISGs required IRF3 and coincided with an ∼10-fold increase in IRF3 from protein stabilization. The induced IRF3 bound to chromatin and localized to the promoters of activated ISGs. While IRF3, STAT1/2, and IRF9 all greatly increased in concentration, there were no corresponding mRNA increases, suggesting that e1a regulates the stabilities of these key activators of innate immune responses, as shown directly for IRF3. Copyright © 2017 Elsevier Inc. All rights reserved.

Membrane-Assisted Growth of DNA Origami Nanostructure Arrays

PubMed Central

2015-01-01

Biological membranes fulfill many important tasks within living organisms. In addition to separating cellular volumes, membranes confine the space available to membrane-associated proteins to two dimensions (2D), which greatly increases their probability to interact with each other and assemble into multiprotein complexes. We here employed two DNA origami structures functionalized with cholesterol moieties as membrane anchors—a three-layered rectangular block and a Y-shaped DNA structure—to mimic membrane-assisted assembly into hierarchical superstructures on supported lipid bilayers and small unilamellar vesicles. As designed, the DNA constructs adhered to the lipid bilayers mediated by the cholesterol anchors and diffused freely in 2D with diffusion coefficients depending on their size and number of cholesterol modifications. Different sets of multimerization oligonucleotides added to bilayer-bound origami block structures induced the growth of either linear polymers or two-dimensional lattices on the membrane. Y-shaped DNA origami structures associated into triskelion homotrimers and further assembled into weakly ordered arrays of hexagons and pentagons, which resembled the geometry of clathrin-coated pits. Our results demonstrate the potential to realize artificial self-assembling systems that mimic the hierarchical formation of polyhedral lattices on cytoplasmic membranes. PMID:25734977

Membrane-assisted growth of DNA origami nanostructure arrays.

PubMed

Kocabey, Samet; Kempter, Susanne; List, Jonathan; Xing, Yongzheng; Bae, Wooli; Schiffels, Daniel; Shih, William M; Simmel, Friedrich C; Liedl, Tim

2015-01-01

Biological membranes fulfill many important tasks within living organisms. In addition to separating cellular volumes, membranes confine the space available to membrane-associated proteins to two dimensions (2D), which greatly increases their probability to interact with each other and assemble into multiprotein complexes. We here employed two DNA origami structures functionalized with cholesterol moieties as membrane anchors--a three-layered rectangular block and a Y-shaped DNA structure--to mimic membrane-assisted assembly into hierarchical superstructures on supported lipid bilayers and small unilamellar vesicles. As designed, the DNA constructs adhered to the lipid bilayers mediated by the cholesterol anchors and diffused freely in 2D with diffusion coefficients depending on their size and number of cholesterol modifications. Different sets of multimerization oligonucleotides added to bilayer-bound origami block structures induced the growth of either linear polymers or two-dimensional lattices on the membrane. Y-shaped DNA origami structures associated into triskelion homotrimers and further assembled into weakly ordered arrays of hexagons and pentagons, which resembled the geometry of clathrin-coated pits. Our results demonstrate the potential to realize artificial self-assembling systems that mimic the hierarchical formation of polyhedral lattices on cytoplasmic membranes.

International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors.

PubMed

Poyner, David R; Sexton, Patrick M; Marshall, Ian; Smith, David M; Quirion, Remi; Born, Walter; Muff, Roman; Fischer, Jan A; Foord, Steven M

2002-06-01

The calcitonin family of peptides comprises calcitonin, amylin, two calcitonin gene-related peptides (CGRPs), and adrenomedullin. The first calcitonin receptor was cloned in 1991. Its pharmacology is complicated by the existence of several splice variants. The receptors for the other members the family are made up of subunits. The calcitonin-like receptor (CL receptor) requires a single transmembrane domain protein, termed receptor activity modifying protein, RAMP1, to function as a CGRP receptor. RAMP2 and -3 enable the same CL receptor to behave as an adrenomedullin receptor. Although the calcitonin receptor does not require RAMP to bind and respond to calcitonin, it can associate with the RAMPs, resulting in a series of receptors that typically have high affinity for amylin and varied affinity for CGRP. This review aims to reconcile what is observed when the receptors are reconstituted in vitro with the properties they show in native cells and tissues. Experimental conditions must be rigorously controlled because different degrees of protein expression may markedly modify pharmacology in such a complex situation. Recommendations, which follow International Union of Pharmacology guidelines, are made for the nomenclature of these multimeric receptors.

The 2-Cys Peroxiredoxin Alkyl Hydroperoxide Reductase C Binds Heme and Participates in Its Intracellular Availability in Streptococcus agalactiae*

PubMed Central

Lechardeur, Delphine; Fernandez, Annabelle; Robert, Bruno; Gaudu, Philippe; Trieu-Cuot, Patrick; Lamberet, Gilles; Gruss, Alexandra

2010-01-01

Heme is a redox-reactive molecule with vital and complex roles in bacterial metabolism, survival, and virulence. However, few intracellular heme partners were identified to date and are not well conserved in bacteria. The opportunistic pathogen Streptococcus agalactiae (group B Streptococcus) is a heme auxotroph, which acquires exogenous heme to activate an aerobic respiratory chain. We identified the alkyl hydroperoxide reductase AhpC, a member of the highly conserved thiol-dependent 2-Cys peroxiredoxins, as a heme-binding protein. AhpC binds hemin with a Kd of 0.5 μm and a 1:1 stoichiometry. Mutagenesis of cysteines revealed that hemin binding is dissociable from catalytic activity and multimerization. AhpC reductase activity was unchanged upon interaction with heme in vitro and in vivo. A group B Streptococcus ahpC mutant displayed attenuation of two heme-dependent functions, respiration and activity of a heterologous catalase, suggesting a role for AhpC in heme intracellular fate. In support of this hypothesis, AhpC-bound hemin was protected from chemical degradation in vitro. Our results reveal for the first time a role for AhpC as a heme-binding protein. PMID:20332091

The Inner Membrane Complex Sub-compartment Proteins Critical for Replication of the Apicomplexan Parasite Toxoplasma gondii Adopt a Pleckstrin Homology Fold*

PubMed Central

Tonkin, Michelle L.; Beck, Josh R.; Bradley, Peter J.; Boulanger, Martin J.

2014-01-01

Toxoplasma gondii, an apicomplexan parasite prevalent in developed nations, infects up to one-third of the human population. The success of this parasite depends on several unique structures including an inner membrane complex (IMC) that lines the interior of the plasma membrane and contains proteins important for gliding motility and replication. Of these proteins, the IMC sub-compartment proteins (ISPs) have recently been shown to play a role in asexual T. gondii daughter cell formation, yet the mechanism is unknown. Complicating mechanistic characterization of the ISPs is a lack of sequence identity with proteins of known structure or function. In support of elucidating the function of ISPs, we first determined the crystal structures of representative members TgISP1 and TgISP3 to a resolution of 2.10 and 2.32 Å, respectively. Structural analysis revealed that both ISPs adopt a pleckstrin homology fold often associated with phospholipid binding or protein-protein interactions. Substitution of basic for hydrophobic residues in the region that overlays with phospholipid binding in related pleckstrin homology domains, however, suggests that ISPs do not retain phospholipid binding activity. Consistent with this observation, biochemical assays revealed no phospholipid binding activity. Interestingly, mapping of conserved surface residues combined with crystal packing analysis indicates that TgISPs have functionally repurposed the phospholipid-binding site likely to coordinate protein partners. Recruitment of larger protein complexes may also be aided through avidity-enhanced interactions resulting from multimerization of the ISPs. Overall, we propose a model where TgISPs recruit protein partners to the IMC to ensure correct progression of daughter cell formation. PMID:24675080

Soybean Ferritin Expression in Saccharomyces cerevisiae Modulates Iron Accumulation and Resistance to Elevated Iron Concentrations

PubMed Central

de Llanos, Rosa; Martínez-Garay, Carlos Andrés; Fita-Torró, Josep; Romero, Antonia María; Martínez-Pastor, María Teresa

2016-01-01

ABSTRACT Fungi, including the yeast Saccharomyces cerevisiae, lack ferritin and use vacuoles as iron storage organelles. This work explored how plant ferritin expression influenced baker's yeast iron metabolism. Soybean seed ferritin H1 (SFerH1) and SFerH2 genes were cloned and expressed in yeast cells. Both soybean ferritins assembled as multimeric complexes, which bound yeast intracellular iron in vivo and, consequently, induced the activation of the genes expressed during iron scarcity. Soybean ferritin protected yeast cells that lacked the Ccc1 vacuolar iron detoxification transporter from toxic iron levels by reducing cellular oxidation, thus allowing growth at high iron concentrations. Interestingly, when simultaneously expressed in ccc1Δ cells, SFerH1 and SFerH2 assembled as heteropolymers, which further increased iron resistance and reduced the oxidative stress produced by excess iron compared to ferritin homopolymer complexes. Finally, soybean ferritin expression led to increased iron accumulation in both wild-type and ccc1Δ yeast cells at certain environmental iron concentrations. IMPORTANCE Iron deficiency is a worldwide nutritional disorder to which women and children are especially vulnerable. A common strategy to combat iron deficiency consists of dietary supplementation with inorganic iron salts, whose bioavailability is very low. Iron-enriched yeasts and cereals are alternative strategies to diminish iron deficiency. Animals and plants possess large ferritin complexes that accumulate, detoxify, or buffer excess cellular iron. However, the yeast Saccharomyces cerevisiae lacks ferritin and uses vacuoles as iron storage organelles. Here, we explored how soybean ferritin expression influenced yeast iron metabolism, confirming that yeasts that express soybean seed ferritin could be explored as a novel strategy to increase dietary iron absorption. PMID:26969708

Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins.

PubMed

Bonuccelli, Gloria; Sotgia, Federica; Schubert, William; Park, David S; Frank, Philippe G; Woodman, Scott E; Insabato, Luigi; Cammer, Michael; Minetti, Carlo; Lisanti, Michael P

2003-10-01

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is absent in the skeletal muscle of DMD patients and mdx mice. At the plasma membrane of skeletal muscle fibers, dystrophin associates with a multimeric protein complex, termed the dystrophin-glycoprotein complex (DGC). Protein members of this complex are normally absent or greatly reduced in dystrophin-deficient skeletal muscle fibers, and are thought to undergo degradation through an unknown pathway. As such, we reasoned that inhibition of the proteasomal degradation pathway might rescue the expression and subcellular localization of dystrophin-associated proteins. To test this hypothesis, we treated mdx mice with the well-characterized proteasomal inhibitor MG-132. First, we locally injected MG-132 into the gastrocnemius muscle, and observed the outcome after 24 hours. Next, we performed systemic treatment using an osmotic pump that allowed us to deliver different concentrations of the proteasomal inhibitor, over an 8-day period. By immunofluorescence and Western blot analysis, we show that administration of the proteasomal inhibitor MG-132 effectively rescues the expression levels and plasma membrane localization of dystrophin, beta-dystroglycan, alpha-dystroglycan, and alpha-sarcoglycan in skeletal muscle fibers from mdx mice. Furthermore, we show that systemic treatment with the proteasomal inhibitor 1) reduces muscle membrane damage, as revealed by vital staining (with Evans blue dye) of the diaphragm and gastrocnemius muscle isolated from treated mdx mice, and 2) ameliorates the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice. Thus, the current study opens new and important avenues in our understanding of the pathogenesis of DMD. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD.

Systematic biochemical characterization of the SAM domains in Eph receptor family from Mus Musculus.

PubMed

Wang, Yue; Li, Qingxia; Zheng, Yunhua; Li, Gang; Liu, Wei

2016-05-13

The Eph receptor family is the largest subfamily of receptor tyrosine kinases and well-known for their pivotal roles in axon guidance, synaptogenesis, artery/venous differentiation and tumorigenesis, etc. Activation of the Eph receptor needs multimerization of the receptors. The intracellular C-terminal SAM domain of Eph receptor was reported to mediate self-association of Eph receptors via the homo SAM-SAM interaction. In this study, we systematically expressed and purified the SAM domain proteins of all fourteen Eph receptors of Mus musculus in Escherichia coli. The FPLC (fast protein liquid chromatography) results showed the recombinant SAM domains were highly homogeneous. Using CD (circular dichroism) spectrometry, we found that the secondary structure of all the SAM domains was typically alpha helical folded and remarkably similar. The thermo-stability tests showed that they were quite stable in solution. SEC-MALS (size exclusion chromatography coupled with multiple angle light scattering) results illustrated 200 μM Eph SAM domains behaved as good monomers in the size-exclusion chromatography. More importantly, DLS (dynamic light scattering) results revealed the overwhelming majority of SAM domains was not multimerized in solution either at 200 μM or 2000 μM protein concentration, which indicating the SAM domain alone was not sufficient to mediate the polymerization of Eph receptor. In summary, our studies provided the systematic biochemical characterizations of the Eph receptor SAM domains and implied their roles in Eph receptor mediated signaling pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

Pathogenic effects of Rift Valley fever virus NSs gene are alleviated in cultured cells by expressed antiviral short hairpin RNAs.

PubMed

Scott, Tristan; Paweska, Janusz T; Arbuthnot, Patrick; Weinberg, Marc S

2012-01-01

Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, may cause severe hepatitis, encephalitis and haemorrhagic fever in humans. There are currently no available licensed vaccines or therapies to treat the viral infection in humans. RNA interference (RNAi)-based viral gene silencing offers a promising approach to inhibiting replication of this highly pathogenic virus. The small (S) segment of the RVFV tripartite genome carries the genetic determinates for pathogenicity during infection. This segment encodes the non-structural S (NSs) and essential nucleocapsid (N) genes. To advance RNAi-based inhibition of RVFV replication, we designed several Pol III short hairpin RNA (shRNA) expression cassettes against the NSs and N genes, including a multimerized plasmid vector that included four shRNA expression cassettes. Effective target silencing was demonstrated using full- and partial-length target reporter assays, and confirmed by western blot analysis of exogenous N and NSs expression. Small RNA northern blots showed detectable RNAi guide strand formation from single and multimerized shRNA constructs. Using a cell culture model of RVFV replication, shRNAs targeting the N gene decreased intracellular nucleocapsid protein concentration and viral replication. The shRNAs directed against the NSs gene reduced NSs protein concentrations and alleviated NSs-mediated cytotoxicity, which may be caused by host transcription suppression. These data are the first demonstration that RNAi activators have a potential therapeutic benefit for countering RVFV infection.

Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity

PubMed Central

Hammerstrom, Troy G.; Horton, Lori B.; Swick, Michelle C.; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M.

2015-01-01

Summary The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthesis operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity, and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His → Asp) and phosphoablative (His → Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (1) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (2) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism. PMID:25402841

Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity.

PubMed

Hammerstrom, Troy G; Horton, Lori B; Swick, Michelle C; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M

2015-02-01

The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO(2)/bicarbonate, and there is a positive correlation between the CO(2)/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His→Asp) and phosphoablative (His→Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism. © 2014 John Wiley & Sons Ltd.

Essential roles of insulin, AMPK signaling and lysyl and prolyl hydroxylases in the biosynthesis and multimerization of adiponectin.

PubMed

Zhang, Lin; Li, Ming-Ming; Corcoran, Marie; Zhang, Shaoping; Cooper, Garth J S

2015-01-05

Post-translational modifications (PTMs) of the adiponectin molecule are essential for its full bioactivity, and defects in PTMs leading to its defective production and multimerization have been linked to the mechanisms of insulin resistance, obesity, and type-2 diabetes. Here we observed that, in differentiated 3T3-L1 adipocytes, decreased insulin signaling caused by blocking of insulin receptors (InsR) with an anti-InsR blocking antibody, increased rates of adiponectin secretion, whereas concomitant elevations in insulin levels counteracted this effect. Adenosine monophosphate-activated protein kinase (AMPK) signaling regulated adiponectin production by modulating the expression of adiponectin receptors, the secretion of adiponectin, and eventually the expression of adiponectin itself. We found that lysyl hydroxylases (LHs) and prolyl hydroxylases (PHs) were expressed in white-adipose tissue of ob/ob mice, wherein LH3 levels were increased compared with controls. In differentiated 3T3-L1 adipocytes, both non-specific inhibition of LHs and PHs by dipyridyl, and specific inhibition of LHs by minoxidil and of P4H with ethyl-3,4-dihydroxybenzoate, caused significant suppression of adiponectin production, more particularly of the higher-order isoforms. Transient gene knock-down of LH3 (Plod3) caused a suppressive effect, especially on the high molecular-weight (HMW) isoforms. These data indicate that PHs and LHs are both required for physiological adiponectin production and in particular are essential for the formation/secretion of the HMW isoforms. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Disturbed secretion of mutant adiponectin associated with the metabolic syndrome.

PubMed

Kishida, Ken; Nagaretani, Hiroyuki; Kondo, Hidehiko; Kobayashi, Hideki; Tanaka, Sachiyo; Maeda, Norikazu; Nagasawa, Azumi; Hibuse, Toshiyuki; Ohashi, Koji; Kumada, Masahiro; Nishizawa, Hitoshi; Okamoto, Yoshihisa; Ouchi, Noriyuki; Maeda, Kazuhisa; Kihara, Shinji; Funahashi, Tohru; Matsuzawa, Yuji

2003-06-20

Adiponectin, an adipocyte-derived protein, consists of collagen-like fibrous and complement C1q-like globular domains, and circulates in human plasma in a multimeric form. The protein exhibits anti-diabetic and anti-atherogenic activities. However, adiponectin plasma concentrations are low in obese subjects, and hypoadiponectinemia is associated with the metabolic syndrome, which is a cluster of insulin resistance, type 2 diabetes mellitus, hypertension, and dyslipidemia. We have recently reported a missense mutation in the adiponectin gene, in which isoleucine at position 164 in the globular domain is substituted with threonine (I164T). Subjects with this mutation showed markedly low level of plasma adiponectin and clinical features of the metabolic syndrome. Here, we examined the molecular characteristics of the mutant protein associated with a genetic cause of hypoadiponectinemia. The current study revealed (1) the mutant protein showed an oligomerization state similar to the wild-type as determined by gel filtration chromatography and, (2) the mutant protein exhibited normal insulin-sensitizing activity, but (3) pulse-chase study showed abnormal secretion of the mutant protein from adipose tissues. Our results suggest that I164T mutation is associated with hypoadiponectinemia through disturbed secretion into plasma, which may contribute to the development of the metabolic syndrome.

Piperazine derivatives inhibit PrP/PrP(res) propagation in vitro and in vivo.

PubMed

Leidel, Fabienne; Eiden, Martin; Geissen, Markus; Hirschberger, Thomas; Tavan, Paul; Giese, Armin; Kretzschmar, Hans A; Schätzl, Hermann; Groschup, Martin H

2014-02-28

Prion diseases are fatal neurodegenerative disorders, which are not curable and no effective treatment exists so far. The major neuropathological change in diseased brains is the conversion of the normal cellular form of the prion protein PrPc(C) into a disease-associated isoform PrP(Sc). PrP(Sc) accumulates into multimeres and fibrillar aggregates, which leads to the formation of amyloid plaques. Increasing evidence indicates a fundamental role of PrP(Sc) species and its aggregation in the pathogenesis of prion diseases, which initiates the pathological cascade and leads to neurodegeneration accompanied by spongiform changes. In search of compounds that have the potential to interfere with PrP(Sc) formation and propagation, we used a cell based assay for the screening of potential aggregation inhibitors. The assay deals with a permanently prion infected cell line that was adapted for a high-throughput screening of a compound library composed of 10,000 compounds (DIVERset 2, ChemBridge). We could detect six different classes of highly potent inhibitors of PrP(Sc) propagation in vitro and identified piperazine derivatives as a new inhibitory lead structure, which increased incubation time of scrapie infected mice. Copyright © 2014 Elsevier Inc. All rights reserved.

Oligomeric Status and Nucleotide Binding Properties of the Plastid ATP/ADP Transporter 1: Toward a Molecular Understanding of the Transport Mechanism

PubMed Central

Deniaud, Aurélien; Panwar, Pankaj; Frelet-Barrand, Annie; Bernaudat, Florent; Juillan-Binard, Céline; Ebel, Christine; Rolland, Norbert; Pebay-Peyroula, Eva

2012-01-01

Background Chloroplast ATP/ADP transporters are essential to energy homeostasis in plant cells. However, their molecular mechanism remains poorly understood, primarily due to the difficulty of producing and purifying functional recombinant forms of these transporters. Methodology/Principal Findings In this work, we describe an expression and purification protocol providing good yields and efficient solubilization of NTT1 protein from Arabidopsis thaliana. By biochemical and biophysical analyses, we identified the best detergent for solubilization and purification of functional proteins, LAPAO. Purified NTT1 was found to accumulate as two independent pools of well folded, stable monomers and dimers. ATP and ADP binding properties were determined, and Pi, a co-substrate of ADP, was confirmed to be essential for nucleotide steady-state transport. Nucleotide binding studies and analysis of NTT1 mutants lead us to suggest the existence of two distinct and probably inter-dependent binding sites. Finally, fusion and deletion experiments demonstrated that the C-terminus of NTT1 is not essential for multimerization, but probably plays a regulatory role, controlling the nucleotide exchange rate. Conclusions/Significance Taken together, these data provide a comprehensive molecular characterization of a chloroplast ATP/ADP transporter. PMID:22438876

Promyelocytic Leukemia Protein (PML) Controls Listeria monocytogenes Infection

PubMed Central

Ribet, David; Lallemand-Breitenbach, Valérie; Ferhi, Omar; Nahori, Marie-Anne; Varet, Hugo

2017-01-01

ABSTRACT The promyelocytic leukemia protein (PML) is the main organizer of stress-responsive subnuclear structures called PML nuclear bodies. These structures recruit multiple interactors and modulate their abundance or their posttranslational modifications, notably by the SUMO ubiquitin-like modifiers. The involvement of PML in antiviral responses is well established. In contrast, the role of PML in bacterial infection remains poorly characterized. Here, we show that PML restricts infection by the pathogenic bacterium Listeria monocytogenes but not by Salmonella enterica serovar Typhimurium. During infection, PML undergoes oxidation-mediated multimerization, associates with the nuclear matrix, and becomes de-SUMOylated due to the pore-forming activity of the Listeria toxin listeriolysin O (LLO). These events trigger an antibacterial response that is not observed during in vitro infection by an LLO-defective Listeria mutant, but which can be phenocopied by specific induction of PML de-SUMOylation. Using transcriptomic and proteomic microarrays, we also characterized a network of immunity genes and cytokines, which are regulated by PML in response to Listeria infection but independently from the listeriolysin O toxin. Our study thus highlights two mechanistically distinct complementary roles of PML in host responses against bacterial infection. PMID:28074026

Inherent flexibility of CLIC6 revealed by crystallographic and solution studies.

PubMed

Ferofontov, Alisa; Strulovich, Roi; Marom, Milit; Giladi, Moshe; Haitin, Yoni

2018-05-02

Chloride intracellular channels (CLICs) are a family of unique proteins, that were suggested to adopt both soluble and membrane-associated forms. Moreover, following this unusual metamorphic change, CLICs were shown to incorporate into membranes and mediate ion conduction in vitro, suggesting multimerization upon membrane insertion. Here, we present a 1.8 Å resolution crystal structure of the CLIC domain of mouse CLIC6 (mCLIC6). The structure reveals a monomeric arrangement and shows a high degree of structural conservation with other CLICs. Small-angle X-ray scattering (SAXS) analysis of mCLIC6 demonstrated that the overall solution structure is similar to the crystallographic conformation. Strikingly, further analysis of the SAXS data using ensemble optimization method unveiled additional elongated conformations, elucidating high structural plasticity as an inherent property of the protein. Moreover, structure-guided perturbation of the inter-domain interface by mutagenesis resulted in a population shift towards elongated conformations of mCLIC6. Additionally, we demonstrate that oxidative conditions induce an increase in mCLIC6 hydrophobicity along with mild oligomerization, which was enhanced by the presence of membrane mimetics. Together, these results provide mechanistic insights into the metamorphic nature of mCLIC6.

Multimeric species in equilibrium in detergent-solubilized Na,K-ATPase.

PubMed

Yoneda, Juliana Sakamoto; Scanavachi, Gustavo; Sebinelli, Heitor Gobbi; Borges, Júlio Cesar; Barbosa, Leandro R S; Ciancaglini, Pietro; Itri, Rosangela

2016-08-01

In this work, we find an equilibrium between different Na,K-ATPase (NKA) oligomeric species solubilized in a non-ionic detergent C12E8 by means of Dynamic Light Scattering (DLS), Analytical Ultracentrifugation (AUC), Small Angle X-ray Scattering (SAXS), Spectrophotometry (absorption at 280/350nm) and enzymatic activity assay. The NKA sample after chromatography purification presented seven different populations as identified by AUC, with monomers and tetramers amounting to ∼55% of the total protein mass in solution. These two species constituted less than 40% of the total protein mass after increasing the NKA concentration. Removal of higher-order oligomer/aggregate species from the NKA solution using 220nm-pore filter resulted in an increase of the specific enzymatic activity. Nevertheless, the enzyme forms new large aggregates over an elapsed time of 20h. The results thus point out that C12E8-solubilized NKA is in a dynamic equilibrium of monomers, tetramers and high-order oligomers/subunit aggregates. These latter have low or null activity. High amount of detergent leads to the dissociation of NKA into smaller aggregates with no enzymatic activity. Copyright © 2016. Published by Elsevier B.V.

Molecular modeling and molecular dynamics simulations based structural analysis of the SG2NA protein variants.

PubMed

Soni, Sangeeta; Tyagi, Chetna; Grover, Abhinav; Goswami, Shyamal K

2014-07-11

SG2NA is a member of the striatin sub-family of WD-40 repeat proteins. Striatin family members have been associated with diverse physiological functions. SG2NA has also been shown to have roles in cell cycle progression, signal transduction etc. They have been known to interact with a number of proteins including Caveolin and Calmodulin and also propagate the formation of a multimeric protein unit called striatin-interacting phosphatase and kinase. As a pre-requisite for such interaction ability, these proteins are known to be unstable and primarily disordered in their arrangement. Earlier we had identified that it has multiple isoforms (namely 35, 78, 87 kDa based on its molecular weight) which are generated by alternative splicing. However, detailed structural information of SG2NA is still eluding the researchers. This study was aimed towards three-dimensional molecular modeling and characterization of SG2NA protein and its isoforms. One structure out of five was selected for each variant having the least value for C score. Out of these, m35 kDa with a C score value of -3.21 was the most poorly determined structure in comparison to m78 kDa and m87 kDa variants with C scores of -1.16 and -1.97 respectively. Further evaluation resulted in about 61.6% residues of m35 kDa, 76.6% residues of m78 kDa and 72.1% residues of m87 kDa falling in the favorable regions of Ramchandran Plot. Molecular dynamics simulations were also carried out to obtain biologically relevant structural models and compared with previous atomic coordinates. N-terminal region of all variants was found to be highly disordered. This study provides first-hand detailed information to understand the structural conformation of SG2NA protein variants (m35 kDa, m78 kDa and m87 kDa). The WD-40 repeat domain was found to constitute antiparallel strands of β-sheets arranged circularly. This study elucidates the crucial structural features of SG2NA proteins which are involved in various protein-protein interactions and also reveals the extent of disorder present in the SG2NA structure crucial for excessive interaction and multimeric protein complexes. The study also potentiates the role of computational approaches for preliminary examination of unknown proteins in the absence of experimental information.

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

Malashkevich, Vladimir N.; Higgins, Chelsea D.; Almo, Steven C.

The coiled-coil is one of the most ubiquitous and well studied protein structural motifs. Significant effort has been devoted to dissecting subtle variations of the typical heptad repeat sequence pattern that can designate larger topological features such as relative α-helical orientation and oligomer size. Here in this paper we report the X-ray structure of a model coiled-coil peptide, HA2-Del-L2seM, which forms an unanticipated core antiparallel dimer with potential sites for discrete higher-order multimerization (trimer or tetramer). In the X-ray structure, a third, partially-ordered α-helix is weakly associated with the antiparallel dimer and analytical ultracentrifugation experiments indicate the peptide forms amore » well-defined tetramer in solution. The HA2-Del-L2seM sequence is closely related to a parent model peptide, HA2-Del, which we previously reported adopts a parallel trimer; HA2-Del-L2seM differs by only hydrophobic leucine to selenomethione mutations and thus this subtle difference is sufficient to switch both relative α-helical topology and number of α-helices participating in the coiled-coil. Comparison of the X-ray structures of HA2-Del-L2seM (reported here) with the HA2-Del parent (reported previously) reveals novel interactions involving the selenomethionine residues that promote antiparallel coiled-coil configuration and preclude parallel trimer formation. Finally, these novel atomic insights are instructive for understanding subtle features that can affect coiled-coil topology and provide additional information for design of antiparallel coiled-coils.« less

Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

PubMed Central

Kim, Dongwook; Yan, Yitang; Valencia, C. Alexander; Liu, Rihe

2012-01-01

Multivalency of targeting ligands provides significantly increased binding strength towards their molecular targets. Here, we report the development of a novel heptameric targeting system, with general applications, constructed by fusing a target-binding domain with the heptamerization domain of the Archaeal RNA binding protein Sm1 through a flexible hinge peptide. The previously reported affibody molecules against EGFR and HER2, ZEGFR and ZHER2, were used as target binding moieties. The fusion molecules were highly expressed in E. coli as soluble proteins and efficiently self-assembled into multimeric targeting ligands with the heptamer as the predominant form. We demonstrated that the heptameric molecules were resistant to protease-mediated digestion or heat- and SDS-induced denaturation. Surface plasmon resonance (SPR) analysis showed that both heptameric ZEGFR and ZHER2 ligands have a significantly enhanced binding strength to their target receptors with a nearly 100 to 1000 fold increase relative to the monomeric ligands. Cellular binding assays showed that heptameric ligands maintained their target-binding specificities similar to the monomeric forms towards their respective receptor. The non-toxic property of each heptameric ligand was demonstrated by the cell proliferation assay. In general,, the heptamerization strategy we describe here could be applied to the facile and efficient engineering of other protein domain- or short peptide-based affinity molecules to acquire significantly improved target-binding strengths with potential applications in the targeted delivery of various imaging or therapeutic agents.. PMID:22912791

A widespread class of reverse transcriptase-related cellular genes.

PubMed

Gladyshev, Eugene A; Arkhipova, Irina R

2011-12-20

Reverse transcriptases (RTs) polymerize DNA on RNA templates. They fall into several structurally related but distinct classes and form an assemblage of RT-like enzymes that, in addition to RTs, also includes certain viral RNA-dependent RNA polymerases (RdRP) synthesizing RNA on RNA templates. It is generally believed that most RT-like enzymes originate from retrotransposons or viruses and have no specific function in the host cell, with telomerases being the only notable exception. Here we report on the discovery and properties of a unique class of RT-related cellular genes collectively named rvt. We present evidence that rvts are not components of retrotransposons or viruses, but single-copy genes with a characteristic domain structure that may contain introns in evolutionarily conserved positions, occur in syntenic regions, and evolve under purifying selection. These genes can be found in all major taxonomic groups including protists, fungi, animals, plants, and even bacteria, although they exhibit patchy phylogenetic distribution in each kingdom. We also show that the RVT protein purified from one of its natural hosts, Neurospora crassa, exists in a multimeric form and has the ability to polymerize NTPs as well as dNTPs in vitro, with a strong preference for NTPs, using Mn(2+) as a cofactor. The existence of a previously unknown class of single-copy RT-related genes calls for reevaluation of the current views on evolution and functional roles of RNA-dependent polymerases in living cells.

Detection of XerC and XerD recombinases in gram-negative bacteria of the family Enterobacteriaceae.

PubMed Central

Sirois, S; Szatmari, G

1995-01-01

XerC and XerD are site-specific recombinases of the lambda integrase family which resolve multimeric replicons to monomers by acting at specific sites such as cer, ckr, nmr, parB, and psi, which are found in plasmids, or at the dif site found in the Escherichia coli chromosome. By using Southern hybridizations to cloned E. coli xerC and xerD genes and a cer-nmr plasmid-based resolution assay, the presence of these genes in several species of Enterobacteriaceae is shown. PMID:7608100

Rationally engineering natural protein assemblies in nanobiotechnology.

PubMed

Howorka, Stefan

2011-08-01

Multimeric protein assemblies are essential components in viruses, bacteria, eukaryotic cells, and organisms where they act as cytoskeletal scaffold, storage containers, or for directional transport. The bottom-up structures can be exploited in nanobiotechnology by harnessing their built-in properties and combining them with new functional modules. This review summarizes the design principles of natural protein assemblies, highlights recent progress in their structural elucidation, and shows how rational engineering can create new biomaterials for applications in vaccine development, biocatalysis, materials science, and synthetic biology. Copyright © 2011 Elsevier Ltd. All rights reserved.

Pentraxin 3 as a Novel Marker in Cardiovascular Diseases?

PubMed Central

Grzesk, Grzegorz; Grzesk, Elzbieta

2011-01-01

Pentraxin 3 (also known as TNFAIP5, TSG-14) belongs to the superfamily of proteins characterized by cyclic multimeric structure. Pentraxin 3 (PTX3) is synthesized locally at the inflammatory sites by endothelial and smooth muscle cells upon exposure to inflammatory signals such as IL-1β, TNF-α or ox-LDL, but not IL-6. Furthermore, PTX3 is highly expressed in vascular cells and myocardial cells in patients with cardiomyopathy. These data suggest that pentraxin 3 may be a useful biomarker for local vascular inflammation and cardiovascular system disorders. PMID:27683398

HIV-1-Specific IgA Monoclonal Antibodies from an HIV-1 Vaccinee Mediate Galactosylceramide Blocking and Phagocytosis

PubMed Central

2018-01-01

ABSTRACT Vaccine-elicited humoral immune responses comprise an array of antibody forms and specificities, with only a fraction contributing to protective host immunity. Elucidation of antibody effector functions responsible for protective immunity against human immunodeficiency virus type 1 (HIV-1) acquisition is a major goal for the HIV-1 vaccine field. Immunoglobulin A (IgA) is an important part of the host defense against pathogens; however, little is known about the role of vaccine-elicited IgA and its capacity to mediate antiviral functions. To identify the antiviral functions of HIV-1-specific IgA elicited by vaccination, we cloned HIV-1 envelope-specific IgA monoclonal antibodies (MAbs) by memory B cell cultures from peripheral blood mononuclear cells from an RV144 vaccinee and produced two IgA clonal cell lines (HG129 and HG130) producing native, nonrecombinant IgA MAbs. The HG129 and HG130 MAbs mediated phagocytosis by monocytes, and HG129 blocked HIV-1 Env glycoprotein binding to galactosylceramide, an alternative HIV-1 receptor. These findings elucidate potential antiviral functions of vaccine-elicited HIV-1 envelope-specific IgA that may act to block HIV-1 acquisition at the portal of entry by preventing HIV-1 binding to galactosylceramide and mediating antibody Fc receptor-mediated virion phagocytosis. Furthermore, these findings highlight the complex and diverse interactions of vaccine-elicited IgA with pathogens that depend on IgA fine specificity and form (e.g., multimeric or monomeric) in the systemic circulation and mucosal compartments. IMPORTANCE Host-pathogen interactions in vivo involve numerous immune mechanisms that can lead to pathogen clearance. Understanding the nature of antiviral immune mechanisms can inform the design of efficacious HIV-1 vaccine strategies. Evidence suggests that both neutralizing and nonneutralizing antibodies can mediate some protection against HIV in animal models. Although numerous studies have characterized the functional properties of HIV-1-specific IgG, more studies are needed on the functional attributes of HIV-1-specific IgA, specifically for vaccine-elicited IgA. Characterization of the functional properties of HIV-1 Env-specific IgA monoclonal antibodies from human vaccine clinical trials are critical toward understanding the capacity of the host immune response to block HIV-1 acquisition. PMID:29321320

Isolated Potato Virus A coat protein possesses unusual properties and forms different short virus-like particles.

PubMed

Ksenofontov, Alexander L; Dobrov, Eugeny N; Fedorova, Natalia V; Serebryakova, Marina V; Prusov, Andrei N; Baratova, Ludmila A; Paalme, Viiu; Järvekülg, Lilian; Shtykova, Eleonora V

2018-05-01

In our previous study, we have observed that the isolated coat proteins (CP) of the Potyvirus Potato Virus A (PVA) virions exhibit an intrinsic tendency to self-associate into various multimeric forms containing some fractions of cross-β-structure. In this report, we studied the effect of solution conditions on the structure and dissociation of isolated PVA CP using a number of complementary physicochemical methods. Analysis of the structure of PVA CP in solution was performed by limited proteolysis with MALDI-TOF mass spectrometry analysis, transmission electron microscopy, intrinsic fluorescence spectroscopy, and synchrotron small angle X-ray scattering (SAXS). Overall structural characteristics of PVA CP obtained by combination of these methods and ab initio shape reconstruction by SAXS show that PVA CP forms large multi-subunit particles. We demonstrate that a mixture of compact virus-like particles (VLP) longer than 30 nm is assembled on dialysis of isolated CP into neutral pH buffer (at low ionic strength). Under conditions of high ionic strength (0.5 M NaCl) and high pH (pH 10.5), PVA dissociates into low compactness oval-shaped particles of approximately 30 subunits (20-30 nm). The results of limited trypsinolysis of these particles (enzyme/substrate ratio 1:100, 30 min) showed the existence of non-cleavable core-fragment, consisting of 137 amino acid residues. Trypsin treatment removed only a short N-terminal fragment in the intact virions. These particles are readily reassembled into regular VLPs by changing pH back to neutral. It is possible that these particles may represent some kind of intermediate in PVA assembly in vitro and in vivo.

Constraints on lateral gene transfer in promoting fimbrial usher protein diversity and function.

PubMed

Stubenrauch, Christopher J; Dougan, Gordon; Lithgow, Trevor; Heinz, Eva

2017-11-01

Fimbriae are long, adhesive structures widespread throughout members of the family Enterobacteriaceae. They are multimeric extrusions, which are moved out of the bacterial cell through an integral outer membrane protein called usher. The complex folding mechanics of the usher protein were recently revealed to be catalysed by the membrane-embedded translocation and assembly module (TAM). Here, we examine the diversity of usher proteins across a wide range of extraintestinal (ExPEC) and enteropathogenic (EPEC) Escherichia coli , and further focus on a so far undescribed chaperone-usher system, with this usher referred to as UshC. The fimbrial system containing UshC is distributed across a discrete set of EPEC types, including model strains like E2348/67, as well as ExPEC ST131, currently the most prominent multi-drug-resistant uropathogenic E. coli strain worldwide. Deletion of the TAM from a naive strain of E. coli results in a drastic time delay in folding of UshC, which can be observed for a protein from EPEC as well as for two introduced proteins from related organisms, Yersinia and Enterobacter We suggest that this models why the TAM machinery is essential for efficient folding of proteins acquired via lateral gene transfer. © 2017 The Authors.

Constraints on lateral gene transfer in promoting fimbrial usher protein diversity and function

PubMed Central

Stubenrauch, Christopher J.; Dougan, Gordon; Lithgow, Trevor

2017-01-01

Fimbriae are long, adhesive structures widespread throughout members of the family Enterobacteriaceae. They are multimeric extrusions, which are moved out of the bacterial cell through an integral outer membrane protein called usher. The complex folding mechanics of the usher protein were recently revealed to be catalysed by the membrane-embedded translocation and assembly module (TAM). Here, we examine the diversity of usher proteins across a wide range of extraintestinal (ExPEC) and enteropathogenic (EPEC) Escherichia coli, and further focus on a so far undescribed chaperone–usher system, with this usher referred to as UshC. The fimbrial system containing UshC is distributed across a discrete set of EPEC types, including model strains like E2348/67, as well as ExPEC ST131, currently the most prominent multi-drug-resistant uropathogenic E. coli strain worldwide. Deletion of the TAM from a naive strain of E. coli results in a drastic time delay in folding of UshC, which can be observed for a protein from EPEC as well as for two introduced proteins from related organisms, Yersinia and Enterobacter. We suggest that this models why the TAM machinery is essential for efficient folding of proteins acquired via lateral gene transfer. PMID:29142104

Zebrafish Meis functions to stabilize Pbx proteins and regulate hindbrain patterning.

PubMed

Waskiewicz, A J; Rikhof, H A; Hernandez, R E; Moens, C B

2001-11-01

Homeodomain-containing Hox proteins regulate segmental identity in Drosophila in concert with two partners known as Extradenticle (Exd) and Homothorax (Hth). These partners are themselves DNA-binding, homeodomain proteins, and probably function by revealing the intrinsic specificity of Hox proteins. Vertebrate orthologs of Exd and Hth, known as Pbx and Meis (named for a myeloid ecotropic leukemia virus integration site), respectively, are encoded by multigene families and are present in multimeric complexes together with vertebrate Hox proteins. Previous results have demonstrated that the zygotically encoded Pbx4/Lazarus (Lzr) protein is required for segmentation of the zebrafish hindbrain and proper expression and function of Hox genes. We demonstrate that Meis functions in the same pathway as Pbx in zebrafish hindbrain development, as expression of a dominant-negative mutant Meis results in phenotypes that are remarkably similar to that of lzr mutants. Surprisingly, expression of Meis protein partially rescues the lzr(-) phenotype. Lzr protein levels are increased in embryos overexpressing Meis and are reduced for lzr mutants that cannot bind to Meis. This implies a mechanism whereby Meis rescues lzr mutants by stabilizing maternally encoded Lzr. Our results define two functions of Meis during zebrafish hindbrain segmentation: that of a DNA-binding partner of Pbx proteins, and that of a post-transcriptional regulator of Pbx protein levels.

Characterization of the zinc-induced Shank3 interactome of mouse synaptosome.

PubMed

Lee, Yeunkum; Ryu, Jae Ryun; Kang, Hyojin; Kim, Yoonhee; Kim, Shinhyun; Zhang, Yinhua; Jin, Chunmei; Cho, Hyo Min; Kim, Won-Ki; Sun, Woong; Han, Kihoon

2017-12-16

Variants of the SHANK3 gene, which encodes a core scaffold protein of the postsynaptic density of excitatory synapses, have been causally associated with numerous brain disorders. Shank3 proteins directly bind zinc ions through their C-terminal sterile α motif domain, which enhances the multimerization and synaptic localization of Shank3, to regulate excitatory synaptic strength. However, no studies have explored whether zinc affects the protein interactions of Shank3, which might contribute to the synaptic changes observed after zinc application. To examine this, we first purified Shank3 protein complexes from mouse brain synaptosomal lysates that were incubated with different concentrations of ZnCl 2 , and analyzed them with mass spectrometry. We used strict criteria to identify 71 proteins that specifically interacted with Shank3 when extra ZnCl 2 was added to the lysate. To characterize the zinc-induced Shank3 interactome, we performed various bioinformatic analyses that revealed significant associations of the interactome with subcellular compartments, including mitochondria, and brain disorders, such as bipolar disorder and schizophrenia. Together, our results showing that zinc affected the Shank3 protein interactions of in vitro mouse synaptosomes provided an additional link between zinc and core synaptic proteins that have been implicated in multiple brain disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

NASA Technical Reports Server (NTRS)

Ji, C.; Chen, Y.; McCarthy, T. L.; Centrella, M.

1999-01-01

Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

Plant-Derived Chimeric Virus Particles for the Diagnosis of Primary Sjögren Syndrome.

PubMed

Tinazzi, Elisa; Merlin, Matilde; Bason, Caterina; Beri, Ruggero; Zampieri, Roberta; Lico, Chiara; Bartoloni, Elena; Puccetti, Antonio; Lunardi, Claudio; Pezzotti, Mario; Avesani, Linda

2015-01-01

Plants are ideal for the production of protein-based nanomaterials because they synthesize and assemble complex multimeric proteins that cannot be expressed efficiently using other platforms. Plant viruses can be thought of as self-replicating proteinaceous nanomaterials generally stable and easily produced in high titers. We used Potato virus X (PVX), chimeric virus particles, and Cowpea mosaic virus, empty virus-like particles to display a linear peptide (lipo) derived from human lipocalin, which is immunodominant in Sjögren's syndrome (SjS) and is thus recognized by autoantibodies in SjS patient serum. These virus-derived nanoparticles were thus used to develop a diagnostic assay for SjS based on a direct enzyme linked immunosorbent assay format. We found that PVX-lipo formulations were more sensitive than the chemically synthesized immunodominant peptide and equally specific when used to distinguish between healthy individuals and SjS patients. Our novel assay therefore allows the diagnosis of SjS using a simple, low-invasive serum test, contrasting with the invasive labial biopsy required for current tests. Our results demonstrate that nanomaterials based on plant viruses can be used as diagnostic reagents for SjS, and could also be developed for the diagnosis of other diseases.

Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase

PubMed Central

Stepper, Peter; Kungulovski, Goran; Jurkowska, Renata Z.; Chandra, Tamir; Krueger, Felix; Reinhardt, Richard

2017-01-01

Abstract DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a–Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20–30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling. PMID:27899645

Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity

DOE PAGES

Hammerstrom, Troy G.; Horton, Lori B.; Swick, Michelle C.; ...

2014-12-30

The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthesis operon. AtxA activity is elevated during growth in media containing glucose and CO 2/bicarbonate, and there is a positive correlation between the CO 2/bicarbonate signal, AtxA activity, and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His → Asp) and phosphoablative (His →more » Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (1) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (2) phosphorylation of H379 in PRD2 disrupts dimer formation. In conclusion, the AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.« less

Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity

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

Hammerstrom, Troy G.; Horton, Lori B.; Swick, Michelle C.

The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthesis operon. AtxA activity is elevated during growth in media containing glucose and CO 2/bicarbonate, and there is a positive correlation between the CO 2/bicarbonate signal, AtxA activity, and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His → Asp) and phosphoablative (His →more » Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (1) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (2) phosphorylation of H379 in PRD2 disrupts dimer formation. In conclusion, the AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.« less

An analog of camptothecin inactive against Topoisomerase I is broadly neutralizing of HIV-1 through inhibition of Vif-dependent APOBEC3G degradation.

PubMed

Bennett, Ryan P; Stewart, Ryan A; Hogan, Priscilla A; Ptak, Roger G; Mankowski, Marie K; Hartman, Tracy L; Buckheit, Robert W; Snyder, Beth A; Salter, Jason D; Morales, Guillermo A; Smith, Harold C

2016-12-01

Camptothecin (CPT) is a natural product discovered to be active against various cancers through its ability to inhibit Topoisomerase I (TOP1). CPT analogs also have anti-HIV-1 (HIV) activity that was previously shown to be independent of TOP1 inhibition. We show that a cancer inactive CPT analog (O2-16) inhibits HIV infection by disrupting multimerization of the HIV protein Vif. Antiviral activity depended on the expression of the cellular viral restriction factor APOBEC3G (A3G) that, in the absence of functional Vif, has the ability to hypermutate HIV proviral DNA during reverse transcription. Our studies demonstrate that O2-16 has low cytotoxicity and inhibits Vif-dependent A3G degradation, enabling A3G packaging into HIV viral particles that results in A3G signature hypermutations in viral genomes. This antiviral activity was A3G-dependent and broadly neutralizing against sixteen HIV clinical isolates from groups M (subtypes A-G), N, and O as well as seven single and multi-drug resistant strains of HIV. Molecular modeling predicted binding near the PPLP motif crucial for Vif multimerization and activity. O2-16 also was active in blocking Vif degradation of APOBEC3F (A3F). We propose that CPT analogs not active against TOP1 have novel therapeutic potential as Vif antagonists that enable A3G-dependent hypermutation of HIV. Copyright © 2016 Elsevier B.V. All rights reserved.

Cold rescue of the thermolabile tailspike intermediate at the junction between productive folding and off-pathway aggregation.

PubMed Central

Betts, S. D.; King, J.

1998-01-01

Off-pathway intermolecular interactions between partially folded polypeptide chains often compete with correct intramolecular interactions, resulting in self-association of folding intermediates into the inclusion body state. Intermediates for both productive folding and off-pathway aggregation of the parallel beta-coil tailspike trimer of phage P22 have been identified in vivo and in vitro using native gel electrophoresis in the cold. Aggregation of folding intermediates was suppressed when refolding was initiated and allowed to proceed for a short period at 0 degrees C prior to warming to 20 degrees C. Yields of refolded tailspike trimers exceeding 80% were obtained using this temperature-shift procedure, first described by Xie and Wetlaufer (1996, Protein Sci 5:517-523). We interpret this as due to stabilization of the thermolabile monomeric intermediate at the junction between productive folding and off-pathway aggregation. Partially folded monomers, a newly identified dimer, and the protrimer folding intermediates were populated in the cold. These species were electrophoretically distinguished from the multimeric intermediates populated on the aggregation pathway. The productive protrimer intermediate is disulfide bonded (Robinson AS, King J, 1997, Nat Struct Biol 4:450-455), while the multimeric aggregation intermediates are not disulfide bonded. The partially folded dimer appears to be a precursor to the disulfide-bonded protrimer. The results support a model in which the junctional partially folded monomeric intermediate acquires resistance to aggregation in the cold by folding further to a conformation that is activated for correct recognition and subunit assembly. PMID:9684883

Structure of FcRY, an avian immunoglobulin receptor related to mammalian mannose receptors, and its complex with IgY

PubMed Central

He, Yongning; Bjorkman, Pamela J.

2011-01-01

Fc receptors transport maternal antibodies across epithelial cell barriers to passively immunize newborns. FcRY, the functional counterpart of mammalian FcRn (a major histocompatibility complex homolog), transfers IgY across the avian yolk sac, and represents a new class of Fc receptor related to the mammalian mannose receptor family. FcRY and FcRn bind immunoglobulins at pH ≤6.5, but not pH ≥7, allowing receptor–ligand association inside intracellular vesicles and release at the pH of blood. We obtained structures of monomeric and dimeric FcRY and an FcRY–IgY complex and explored FcRY's pH-dependent binding mechanism using electron cryomicroscopy (cryoEM) and small-angle X-ray scattering. The cryoEM structure of FcRY at pH 6 revealed a compact double-ring “head,” in which the N-terminal cysteine-rich and fibronectin II domains were folded back to contact C-type lectin-like domains 1–6, and a “tail” comprising C-type lectin-like domains 7–8. Conformational changes at pH 8 created a more elongated structure that cannot bind IgY. CryoEM reconstruction of FcRY dimers at pH 6 and small-angle X-ray scattering analysis at both pH values confirmed both structures. The cryoEM structure of the FcRY–IgY revealed symmetric binding of two FcRY heads to the dimeric FcY, each head contacting the CH4 domain of one FcY chain. FcRY shares structural properties with mannose receptor family members, including a head and tail domain organization, multimerization that may regulate ligand binding, and pH-dependent conformational changes. Our results facilitate understanding of immune recognition by the structurally related mannose receptor family and comparison of diverse methods of Ig transport across evolution. PMID:21746914

Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex.

PubMed

Kyrchanova, Olga; Mogila, Vladic; Wolle, Daniel; Deshpande, Girish; Parshikov, Alexander; Cléard, Fabienne; Karch, Francois; Schedl, Paul; Georgiev, Pavel

2016-07-01

Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex

PubMed Central

Wolle, Daniel; Deshpande, Girish; Parshikov, Alexander; Cléard, Fabienne; Karch, Francois; Schedl, Paul; Georgiev, Pavel

2016-01-01

Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors. PMID:27428541

Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking.

PubMed

Mayor, S; Rothberg, K G; Maxfield, F R

1994-06-24

Glycosyl-phosphatidylinositol (GPI)-anchored proteins have been reported to reside in clusters collected over small membrane invaginations called caveolae. The detection of different GPI-anchored proteins with fluorescently labeled monoclonal antibodies showed that these proteins are not constitutively concentrated in caveolae; they enter these structures independently after cross-linking with polyclonal secondary antibodies. Analysis of the cell surface distribution of the GPI-anchored folate receptor by electron microscopy confirms these observations. Thus, multimerization of GPI-anchored proteins regulates their sequestration in caveolae, but in the absence of agents that promote clustering they are diffusely distributed over the plasma membrane.

Functional Sub-states by High-pressure Macromolecular Crystallography.

PubMed

Dhaussy, Anne-Claire; Girard, Eric

2015-01-01

At the molecular level, high-pressure perturbation is of particular interest for biological studies as it allows trapping conformational substates. Moreover, within the context of high-pressure adaptation of deep-sea organisms, it allows to decipher the molecular determinants of piezophily. To provide an accurate description of structural changes produced by pressure in a macromolecular system, developments have been made to adapt macromolecular crystallography to high-pressure studies. The present chapter is an overview of results obtained so far using high-pressure macromolecular techniques, from nucleic acids to virus capsid through monomeric as well as multimeric proteins.

Phosphinic acid functionalized polyazacycloalkane chelators for radiodiagnostics and radiotherapeutics: unique characteristics and applications.

PubMed

Notni, Johannes; Šimeček, Jakub; Wester, Hans-Jürgen

2014-06-01

Given the wide application of positron emission tomography (PET), positron-emitting metal radionuclides have received much attention recently. Of these, gallium-68 has become particularly popular, as it is the only PET nuclide commercially available from radionuclide generators, therefore allowing local production of PET radiotracers independent of an on-site cyclotron. Hence, interest in optimized bifunctional chelators for the elaboration of (68) Ga-labeled bioconjugates has been rekindled as well, resulting in the development of improved triazacyclononane-triphosphinate (TRAP) ligand structures. The most remarkable features of these ligands are unparalleled selectivity for Ga(III) , rapid Ga(III) complexation kinetics, extraordinarily high thermodynamic stability, and kinetic inertness of the respective Ga(III) chelates. As a result, TRAP chelators exhibit very favorable (68) Ga-labeling properties. Based on the scaffolds NOPO (1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)phosphinic acid]-7-[methylene(2-carboxyethyl)phosphinic acid]) and TRAP-Pr, tailored for convenient preparation of (68) Ga-labeled monomeric and multimeric bioconjugates, a variety of novel (68) Ga radiopharmaceuticals have been synthesized. These include bisphosphonates, somatostatin receptor ligands, prostate-specific membrane antigen (PSMA)-targeting peptides, and cyclic RGD pentapeptides, for in vivo PET imaging of bone, neuroendocrine tumors, prostate cancer, and integrin expression, respectively. Furthermore, TRAP-based (68) Ga-labeled gadolinium(III) complexes have been proposed as bimodal probes for PET/MRI, and a cyclen-based analogue of TRAP-Pr has been suggested for the elaboration of targeted radiotherapeutics comprising radiolanthanide ions. Thus, polyazacycloalkane-based polyphosphinic acid chelators are a powerful toolbox for pharmaceutical research, particularly for the development of (68) Ga radiopharmaceuticals. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Roles of RIG-I N-terminal tandem CARD and splice variant in TRIM25-mediated antiviral signal transduction

PubMed Central

Gack, Michaela U.; Kirchhofer, Axel; Shin, Young C.; Inn, Kyung-Soo; Liang, Chengyu; Cui, Sheng; Myong, Sua; Ha, Taekjip; Hopfner, Karl-Peter; Jung, Jae U.

2008-01-01

The caspase recruitment domain (CARD) of intracellular adaptors and sensors plays a critical role in the assembly of signaling complexes involved in innate host defense against pathogens and in the regulation of inflammatory responses. The cytosolic receptor retinoic acid-inducible gene-I (RIG-I) recognizes viral RNA in a 5′-triphosphate-dependent manner and initiates an antiviral signaling cascade. Upon viral infection, the N-terminal CARDs of RIG-I undergo the K63-linked ubiquitination induced by tripartite motif protein 25 (TRIM25), critical for the interaction of RIG-I with its downstream signaling partner MAVS/VISA/IPS-1/Cardif. Here, we demonstrate the distinct roles of RIG-I first and second CARD in TRIM25-mediated RIG-I ubiquitination: TRIM25 binds the RIG-I first CARD and subsequently ubiquitinates its second CARD. The T55I mutation in RIG-I first CARD abolishes TRIM25 interaction, whereas the K172R mutation in the second CARD eliminates polyubiquitin attachment. The necessity of the intact tandem CARD for RIG-I function is further evidenced by a RIG-I splice variant (SV) whose expression is robustly up-regulated upon viral infection. The RIG-I SV carries a short deletion (amino acids 36–80) within the first CARD and thereby loses TRIM25 binding, CARD ubiquitination, and downstream signaling ability. Furthermore, because of its robust inhibition of virus-induced RIG-I multimerization and RIG-I-MAVS signaling complex formation, this SV effectively suppresses the RIG-I-mediated IFN-β production. This study not only elucidates the vital role of the intact tandem CARD for TRIM25-mediated RIG-I activation but also identifies the RIG-I SV as an off-switch regulator of its own signaling pathway. PMID:18948594

Roles of RIG-I N-terminal tandem CARD and splice variant in TRIM25-mediated antiviral signal transduction.

PubMed

Gack, Michaela U; Kirchhofer, Axel; Shin, Young C; Inn, Kyung-Soo; Liang, Chengyu; Cui, Sheng; Myong, Sua; Ha, Taekjip; Hopfner, Karl-Peter; Jung, Jae U

2008-10-28

The caspase recruitment domain (CARD) of intracellular adaptors and sensors plays a critical role in the assembly of signaling complexes involved in innate host defense against pathogens and in the regulation of inflammatory responses. The cytosolic receptor retinoic acid-inducible gene-I (RIG-I) recognizes viral RNA in a 5'-triphosphate-dependent manner and initiates an antiviral signaling cascade. Upon viral infection, the N-terminal CARDs of RIG-I undergo the K(63)-linked ubiquitination induced by tripartite motif protein 25 (TRIM25), critical for the interaction of RIG-I with its downstream signaling partner MAVS/VISA/IPS-1/Cardif. Here, we demonstrate the distinct roles of RIG-I first and second CARD in TRIM25-mediated RIG-I ubiquitination: TRIM25 binds the RIG-I first CARD and subsequently ubiquitinates its second CARD. The T(55)I mutation in RIG-I first CARD abolishes TRIM25 interaction, whereas the K(172)R mutation in the second CARD eliminates polyubiquitin attachment. The necessity of the intact tandem CARD for RIG-I function is further evidenced by a RIG-I splice variant (SV) whose expression is robustly up-regulated upon viral infection. The RIG-I SV carries a short deletion (amino acids 36-80) within the first CARD and thereby loses TRIM25 binding, CARD ubiquitination, and downstream signaling ability. Furthermore, because of its robust inhibition of virus-induced RIG-I multimerization and RIG-I-MAVS signaling complex formation, this SV effectively suppresses the RIG-I-mediated IFN-beta production. This study not only elucidates the vital role of the intact tandem CARD for TRIM25-mediated RIG-I activation but also identifies the RIG-I SV as an off-switch regulator of its own signaling pathway.

Erythroid Kruppel-like factor (EKLF) is recruited to the γ-globin gene promoter as a co-activator and is required for γ-globin gene induction by short-chain fatty acid derivatives

PubMed Central

Perrine, Susan P.; Mankidy, Rishikesh; Boosalis, Michael S.; Bieker, James J.; Faller, Douglas V.

2011-01-01

Objectives The erythroid Kruppel-like factor (EKLF) is an essential transcription factor for β-type globin gene switching, and specifically activates transcription of the adult β-globin gene promoter. We sought to determine if EKLF is also required for activation of the γ-globin gene by short-chain fatty acid (SCFA) derivatives, which are now entering clinical trials. Methods The functional and physical interaction of EKLF and co-regulatory molecules with the endogenous human globin gene promoters was studied in primary human erythroid progenitors and cell lines, using chromatin immunoprecipitation (ChIP) assays and genetic manipulation of the levels of EKLF and co-regulators. Results and conclusions Knockdown of EKLF prevents SCFA-induced expression of the γ-globin promoter in a stably expressed μLCRβprRlucAγprFluc cassette, and prevents induction of the endogenous γ-globin gene in primary human erythroid progenitors. EKLF is actively recruited to endogenous γ-globin gene promoters after exposure of primary human erythroid progenitors, and murine hematopoietic cell lines, to SCFA derivatives. The core ATPase BRG1 subunit of the human SWI/WNF complex, a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure, is also required for γ-globin gene induction by SCFA derivatives. BRG1 is actively recruited to the endogenous γ-globin promoter of primary human erythroid progenitors by exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes. PMID:19220418

CaMKII in the Cardiovascular System: Sensing Redox States

PubMed Central

Erickson, Jeffrey R.; He, B. Julie; Grumbach, Isabella M.; Anderson, Mark E

2013-01-01

The multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) is now recognized to play a central role in pathological events in the cardiovascular system. CaMKII has diverse downstream targets that promote vascular disease, heart failure and arrhythmias, so improved understanding of CaMKII signaling has the potential to lead to new therapies for cardiovascular disease. CaMKII is a multimeric serine-threonine kinase that is initially activated by binding calcified calmodulin (Ca2+/CaM). Under conditions of sustained exposure to elevated Ca2+/CaM CaMKII transitions into a Ca2+/CaM-autonomous enzyme by two distinct but parallel processes. Autophosphorylation of threonine 287 in the CaMKII regulatory domain ‘traps’ CaMKII into an open configuration even after Ca2+/CaM unbinding. More recently, our group identified a pair of methionines (281/282) in the CaMKII regulatory domain that undergo a partially reversible oxidation which, like autophosphorylation, prevents CaMKII from inactivating after Ca2+/CaM unbinding. Here we review roles of CaMKII in cardiovascular disease with an eye to understanding how CaMKII may act as a transduction signal to connect pro-oxidant conditions into specific downstream pathological effects that are relevant to rare and common forms of cardiovascular disease. PMID:21742790

Grb7 protein RA domain oligomerization.

PubMed

Godamudunage, Malika P; Foster, Albert; Warren, Darius; Lyons, Barbara A

2017-08-01

The growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is often coamplified with the erythroblastosis oncogene B 2 receptor in 20% to 30% of breast cancer patients. Grb7 overexpression has been linked to increased cell migration and cancer metastasis. The ras associating and pleckstrin homology domain region of Grb7 has been reported to interact with various other downstream signaling proteins such as four and half Lin11, Isl-1, Mec-3 (LIM) domains isoform 2 and filamin α. These interactions are believed to play a role in regulating Grb7-mediated cell migration function. The full-length Grb7 protein has been shown to dimerize, and the oligomeric state of the Grb7SH2 domain has been extensively studied; however, the oligomerization state of the ras associating and pleckstrin homology domains, and the importance of this oligomerization in Grb7 function, is yet to be fully known. In this study, we characterize the oligomeric state of the Grb7RA domain using size exclusion chromatography, nuclear magnetic resonance, nuclear relaxation studies, glutaraldehyde cross linking, and dynamic light scattering. We report the Grb7RA domain can exist in transient multimeric forms and, based upon modeling results, postulate the potential role of Grb7RA domain oligomerization in Grb7 function. Copyright © 2017 John Wiley & Sons, Ltd.

Isolation and quantification of Quillaja saponaria Molina saponins and lipids in iscom-matrix and iscoms.

PubMed

Behboudi, S; Morein, B; Rönnberg, B

1995-12-01

In the iscom, multiple copies of antigen are attached by hydrophobic interaction to a matrix which is built up by Quillaja triterpenoid saponins and lipids. Thus, the iscom presents antigen in multimeric form in a small particle with a built-in adjuvant resulting in a highly immunogenic antigen formulation. We have designed a chloroform-methanol-water extraction procedure to isolate the triterpenoid saponins and lipids incorporated into iscom-matrix and iscoms. The triterpenoids in the triterpenoid phase were quantitated using orcinol sulfuric acid detecting their carbohydrate chains and by HPLC. The cholesterol and phosphatidylcholine in the lipid phase were quantitated by HPLC and a commercial colorimetric method for the cholesterol. The quantitative methods showed an almost total separation and recovery of triterpenoids and lipids in their respective phases, while protein was detected in all phases after extraction. The protein content was determined by the method of Lowry and by amino acid analysis. Amino acid analysis was shown to be the reliable method of the two to quantitate proteins in iscoms. In conclusion, simple, reproducible and efficient procedures have been designed to isolate and quantitate the triterpenoids and lipids added for preparation of iscom-matrix and iscoms. The procedures described should also be useful to adequately define constituents in prospective vaccines.

Subcellular Localization and Rolling Circle Replication of Peach Latent Mosaic Viroid: Hallmarks of Group A Viroids

PubMed Central

Bussière, F.; Lehoux, J.; Thompson, D. A.; Skrzeczkowski, L. J.; Perreault, J.-P.

1999-01-01

We characterized the peach latent mosaic viroid (PLMVd) replication intermediates that accumulate in infected peach leaves and determined the tissue and subcellular localization of the RNA species. Using in situ hybridization, we showed that PLMVd strands of both plus and minus polarities concentrate in the cells forming the palisade parenchyma. At the cellular level, PLMVd was found to accumulate predominantly in chloroplasts. Northern blot analyses demonstrated that PLMVd replicates via a symmetric mode involving the accumulation of both circular and linear monomeric strands of both polarities. No multimeric conformer was detected, indicating that both strands self-cleave efficiently via their hammerhead sequences. Dot blot hybridizations revealed that PLMVd strands of both polarities accumulate equally but that the relative concentrations vary by more than 50-fold between peach cultivars. Taken together these results establish two hallmarks for the classification of viroids. Group A viroids (e.g., PLMVd), which possess hammerhead structures, replicate in the chloroplasts via the symmetric mode. By contrast, group B viroids, which share a conserved central region, replicate in the nucleus via an asymmetric mechanism. This is an important difference between self-cleaving and non-self-cleaving viroids, and the implications for the evolutionary origin and replication are discussed. PMID:10400727

Independent Neuronal Origin of Seizures and Behavioral Comorbidities in an Animal Model of a Severe Childhood Genetic Epileptic Encephalopathy

PubMed Central

Asinof, Samuel K.; Sukoff Rizzo, Stacey J.; Buckley, Alexandra R.; Beyer, Barbara J.; Letts, Verity A.; Frankel, Wayne N.; Boumil, Rebecca M.

2015-01-01

The childhood epileptic encephalopathies (EE’s) are seizure disorders that broadly impact development including cognitive, sensory and motor progress with severe consequences and comorbidities. Recently, mutations in DNM1 (dynamin 1) have been implicated in two EE syndromes, Lennox-Gastaut Syndrome and Infantile Spasms. Dnm1 encodes dynamin 1, a large multimeric GTPase necessary for activity-dependent membrane recycling in neurons, including synaptic vesicle endocytosis. Dnm1Ftfl or “fitful” mice carry a spontaneous mutation in the mouse ortholog of DNM1 and recapitulate many of the disease features associated with human DNM1 patients, providing a relevant disease model of human EE’s. In order to examine the cellular etiology of seizures and behavioral and neurological comorbidities, we engineered a conditional Dnm1Ftfl mouse model of DNM1 EE. Observations of Dnm1 Ftfl/flox mice in combination with various neuronal subpopulation specific cre strains demonstrate unique seizure phenotypes and clear separation of major neurobehavioral comorbidities from severe seizures associated with the germline model. This demonstration of pleiotropy suggests that treating seizures per se may not prevent severe comorbidity observed in EE associated with dynamin-1 mutations, and is likely to have implications for other genetic forms of EE. PMID:26125563

Molecular Dynamics Simulation of the Antiamoebin Ion Channel: Linking Structure and Conductance

NASA Technical Reports Server (NTRS)

Wilson, Michael A.; Wei, Chenyu; Bjelkmar, Paer; Wallace, B. A.; Pohorille, Andrew

2011-01-01

Molecular dynamics simulations were carried out in order to ascertain which of the potential multimeric forms of the transmembrane peptaibol channel, antiamoebin, is consistant with its measured conductance. Estimates of the conductance obtained through counting ions that cross the channel and by solving the Nernst-Planck equation yield consistent results, indicating that the motion of ions inside the channel can be satisfactorily described as diffusive.The calculated conductance of octameric channels is markedly higher than the conductance measured in single channel recordings, whereas the tetramer appears to be non-conducting. The conductance of the hexamer was estimated to be 115+/-34 pS and 74+/-20 pS, at 150 mV and 75 mV, respectively, in satisfactory agreement with the value of 90 pS measured at 75 mV. On this basis we propose that the antiamoebin channel consists of six monomers. Its pore is large enough to accommodate K(+) and Cl(-) with their first solvation shells intact. The free energy barrier encountered by K(+) is only 2.2 kcal/mol whereas Cl(-) encounters a substantially higher barrier of nearly 5 kcal/mol. This difference makes the channel selective for cations. Ion crossing events are shown to be uncorrelated and follow Poisson statistics. keywords: ion channels, peptaibols, channel conductance, molecular dynamics

Effects of different isoforms of apoE on aggregation of the α-synuclein protein implicated in Parkinson's disease.

PubMed

Emamzadeh, Fatemeh Nouri; Aojula, Harmesh; McHugh, Patrick C; Allsop, David

2016-04-08

Parkinson's disease is a progressive brain disorder due to the degeneration of dopaminergic neurons in the substantia nigra. The accumulation of aggregated forms of α-synuclein protein into Lewy bodies is one of the characteristic features of this disease although the pathological role of any such protein deposits in causing neurodegeneration remains elusive. Here, the effects of different apolipoprotein E isoforms (apoE2, apoE3, apoE4) on the aggregation of α-synuclein in vitro were examined using thioflavin T assays and also an immunoassay to detect the formation of multimeric forms. Our results revealed that the aggregation of α-synuclein is influenced by apoE concentration. At low concentrations of apoE (<15nM), all of the isoforms were able to increase the aggregation of α-synuclein (50μM), with apoE4 showing the greatest stimulatory effect. This is in contrast to a higher concentration (>15nM) of these isoforms, where a decrease in the aggregation of α-synuclein was noted. The data show that exceptionally low levels of apoE may seed α-syn aggregation, which could potentially lead to the pathogenesis of α-synuclein-induced neurodegeneration. On the other hand, higher levels of apoE could potentially lower the degree of α-synuclein aggregation and confer protection. The differential effects noted with apoE4 could explain why this particular isoform results in an earlier age of onset for Parkinson's disease. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Mammalian amyloidogenic proteins promote prion nucleation in yeast.

PubMed

Chandramowlishwaran, Pavithra; Sun, Meng; Casey, Kristin L; Romanyuk, Andrey V; Grizel, Anastasiya V; Sopova, Julia V; Rubel, Aleksandr A; Nussbaum-Krammer, Carmen; Vorberg, Ina M; Chernoff, Yury O

2018-03-02

Fibrous cross-β aggregates (amyloids) and their transmissible forms (prions) cause diseases in mammals (including humans) and control heritable traits in yeast. Initial nucleation of a yeast prion by transiently overproduced prion-forming protein or its (typically, QN-rich) prion domain is efficient only in the presence of another aggregated (in most cases, QN-rich) protein. Here, we demonstrate that a fusion of the prion domain of yeast protein Sup35 to some non-QN-rich mammalian proteins, associated with amyloid diseases, promotes nucleation of Sup35 prions in the absence of pre-existing aggregates. In contrast, both a fusion of the Sup35 prion domain to a multimeric non-amyloidogenic protein and the expression of a mammalian amyloidogenic protein that is not fused to the Sup35 prion domain failed to promote prion nucleation, further indicating that physical linkage of a mammalian amyloidogenic protein to the prion domain of a yeast protein is required for the nucleation of a yeast prion. Biochemical and cytological approaches confirmed the nucleation of protein aggregates in the yeast cell. Sequence alterations antagonizing or enhancing amyloidogenicity of human amyloid-β (associated with Alzheimer's disease) and mouse prion protein (associated with prion diseases), respectively, antagonized or enhanced nucleation of a yeast prion by these proteins. The yeast-based prion nucleation assay, developed in our work, can be employed for mutational dissection of amyloidogenic proteins. We anticipate that it will aid in the identification of chemicals that influence initial amyloid nucleation and in searching for new amyloidogenic proteins in a variety of proteomes. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

An automated graphics tool for comparative genomics: the Coulson plot generator

PubMed Central

2013-01-01

Background Comparative analysis is an essential component to biology. When applied to genomics for example, analysis may require comparisons between the predicted presence and absence of genes in a group of genomes under consideration. Frequently, genes can be grouped into small categories based on functional criteria, for example membership of a multimeric complex, participation in a metabolic or signaling pathway or shared sequence features and/or paralogy. These patterns of retention and loss are highly informative for the prediction of function, and hence possible biological context, and can provide great insights into the evolutionary history of cellular functions. However, representation of such information in a standard spreadsheet is a poor visual means from which to extract patterns within a dataset. Results We devised the Coulson Plot, a new graphical representation that exploits a matrix of pie charts to display comparative genomics data. Each pie is used to describe a complex or process from a separate taxon, and is divided into sectors corresponding to the number of proteins (subunits) in a complex/process. The predicted presence or absence of proteins in each complex are delineated by occupancy of a given sector; this format is visually highly accessible and makes pattern recognition rapid and reliable. A key to the identity of each subunit, plus hierarchical naming of taxa and coloring are included. A java-based application, the Coulson plot generator (CPG) automates graphic production, with a tab or comma-delineated text file as input and generating an editable portable document format or svg file. Conclusions CPG software may be used to rapidly convert spreadsheet data to a graphical matrix pie chart format. The representation essentially retains all of the information from the spreadsheet but presents a graphically rich format making comparisons and identification of patterns significantly clearer. While the Coulson plot format is highly useful in comparative genomics, its original purpose, the software can be used to visualize any dataset where entity occupancy is compared between different classes. Availability CPG software is available at sourceforge http://sourceforge.net/projects/coulson and http://dl.dropbox.com/u/6701906/Web/Sites/Labsite/CPG.html PMID:23621955

Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges

PubMed Central

Lakhashe, Samir K.; Velu, Vijayakumar; Sciaranghella, Gaia; Siddappa, Nagadenahalli B.; DiPasquale, Janet M.; Hemashettar, Girish; Yoon, John K.; Rasmussen, Robert A.; Yang, Feng; Lee, Sandra J.; Montefiori, David C.; Novembre, Francis J.; Villinger, François; Amara, Rama Rao; Kahn, Maria; Hu, Shiu-Lok; Li, Sufen; Li, Zhongxia; Frankel, Fred R.; Robert-Guroff, Marjorie; Johnson, Welkin E.; Lieberman, Judy; Ruprecht, Ruth M.

2011-01-01

We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus >90%; these RM also had strong SIV Gag-specific proliferation of CD8+ T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the α4β7-expressing CD4+ T cells; the latter have been implicated as preferential virus targets in-vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection. PMID:21693155

Unconjugated Bilirubin Inhibits Proteolytic Cleavage of von Willebrand Factor by ADAMTS13 Protease

PubMed Central

Lu, Rui-Nan; Yang, Shangbin; Wu, Haifeng M.; Zheng, X. Long

2015-01-01

Summary Background Bilirubin is a yellow breakdown product of heme catabolism. Increased serum levels of unconjugated bilirubin are conditions commonly seen in premature neonates and adults with acute hemolysis including thrombotic microangiopathy. Previous studies have shown that unconjugated bilirubin lowers plasma ADAMTS13 activity, but the mechanism is not fully understood. Objectives The study is to determine whether unconjugated bilirubin directly inhibits the cleavage of von Willebrand factor (VWF) and its analogs by ADAMTS13. Methods Fluorogenic, SELDI-TOF mass spectrometric assay, and Western blotting analyses were employed to address this question. Results Unconjugated bilirubin inhibits the cleavage of F485-rVWF73-H, D633-rVWF73-H, and GST-rVWF71-11K by ADAMTS13 in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of ~13 μM, ~70 μM, and ~17 μM, respectively. Unconjugated bilirubin also dose-dependently inhibits the cleavage of multimeric VWF by ADAMTS13 under denaturing conditions. The inhibitory activity of bilirubin on the cleavage of D633-rVWF73-H and multimeric VWF, but not F485-rVWF73-H, was eliminated after incubation with bilirubin oxidase that converts bilirubin to biliverdin. Furthermore, plasma ADAMTS13 activity in patients with hyperbilirubinemia is lower prior to than after treatment with bilirubin oxidase. Conclusions unconjugated bilirubin directly inhibits ADAMTS13’s ability to cleave both peptidyl and native VWF substrates in addition to its interference with certain fluorogenic assays. Our findings may help proper interpretation of ADAMTS13 results under pathological conditions. Whether elevated serum unconjugated bilirubin has an adverse effect in vivo remains to be determined in our future study. PMID:25782102

Endothelin-1 stimulates catalase activity through the PKCδ mediated phosphorylation of Serine 167

PubMed Central

Rafikov, Ruslan; Kumar, Sanjiv; Aggarwal, Saurabh; Hou, Yali; Kangath, Archana; Pardo, Daniel; Fineman, Jeffrey R.; Black, Stephen M.

2013-01-01

Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells (PAEC) to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H2O2) levels. These changes correlated with an increase in serine phosphorylated catalase. Using the inhibitory peptide δV1.1, this phosphorylation was shown to be PKCδ dependent. Mass spectrometry identified serine167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from E.coli or transiently transfected COS-7 cells, demonstrated that S167D-catalase had an increased ability to degrade H2O2 compared to the wildtype enzyme. Using a phospho-specific antibody, we were able to verify that pS167 catalase levels are modulated in lambs with acute increases in PBF in the presence and absence of the ET receptor antagonist, tezosentan. S167 is being located on the dimeric interface suggesting it could be involved in regulating the formation of catalase tetramers. To evaluate this possibility we utilized analytical gel-filtration to examine the multimeric structure of recombinant wildtype- and S167D-catalase. We found that recombinant wildtype catalase was present as a mixture of monomers and dimers while S167D catalase was primarily tetrameric. Further, the incubation of wildtype catalase with PKCδ was sufficient to convert wildtype catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity. PMID:24211614

The X-ray Crystal Structure of the Phage Tail Terminator Protein Reveals the Biologically Relevant Hexameric Rang Structure and Demonstrates a Conserved mechanism of Tail Termination among Divrse Long Tailed Phages

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

Pell, L.; Liu, A; Edmonds, L

The tail terminator protein (TrP) plays an essential role in phage tail assembly by capping the rapidly polymerizing tail once it has reached its requisite length and serving as the interaction surface for phage heads. Here, we present the 2.7-A crystal structure of a hexameric ring of gpU, the TrP of phage ?. Using sequence alignment analysis and site-directed mutagenesis, we have shown that this multimeric structure is biologically relevant and we have delineated its functional surfaces. Comparison of the hexameric crystal structure with the solution structure of gpU that we previously solved using NMR spectroscopy shows large structural changesmore » occurring upon multimerization and suggests a mechanism that allows gpU to remain monomeric at high concentrations on its own, yet polymerize readily upon contact with an assembled tail tube. The gpU hexamer displays several flexible loops that play key roles in head and tail binding, implying a role for disorder-to-order transitions in controlling assembly as has been observed with other ? morphogenetic proteins. Finally, we have found that the hexameric structure of gpU is very similar to the structure of a putative TrP from a contractile phage tail even though it displays no detectable sequence similarity. This finding coupled with further bioinformatic investigations has led us to conclude that the TrPs of non-contractile-tailed phages, such as ?, are evolutionarily related to those of contractile-tailed phages, such as P2 and Mu, and that all long-tailed phages may utilize a conserved mechanism for tail termination.« less

Regulation of protein multipoint adsorption on ion-exchange adsorbent and its application to the purification of macromolecules.

PubMed

Huang, Yongdong; Bi, Jingxiu; Zhao, Lan; Ma, Guanghui; Su, Zhiguo

2010-12-01

Ion-exchange chromatography (IEC) using commercial ionic absorbents is a widely used technique for protein purification. Protein adsorption onto ion-exchange adsorbents often involves a multipoint adsorption. In IEC of multimeric proteins or "soft" proteins, the intense multipoint binding would make the further desorption difficult, even lead to the destruction of protein structure and the loss of its biological activity. In this paper, DEAE Sepharose FF adsorbents with controllable ligand densities from 0.020 to 0.183 mmol/ml were synthesized, and then the effect of ligand density on the static ion-exchange adsorption of bovine serum albumin (BSA) onto DEAE Sepharose FF was studied by batch adsorption technique. Steric mass-action (SMA) model was employed to analyze the static adsorption behavior. The results showed that the SMA model parameters, equilibrium constant (K(a)), characteristic number of binding sites (υ) and steric factor (σ), increased gradually with ligand density. Thus, it was feasible to regulate BSA multipoint adsorption by modulating the ligand density of ion-exchange adsorbent. Furthermore, IEC of hepatitis B surface antigen (HBsAg) using DEAE Sepharose FF adsorbents with different ligand densities was carried out, and the activity recovery of HBsAg was improved from 42% to 67% when the ligand density was decreased from 0.183 to 0.020 mmol/ml. Taking the activity recovery of HBsAg, the purification factor and the binding capacity into account, DEAE Sepharose FF with a ligand density of 0.041 mmol/ml was most effective for the purification of HBsAg. Such a strategy may also be beneficial for the purification of macromolecules and multimeric proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

A Restricted Repertoire of De Novo Mutations in ITPR1 Cause Gillespie Syndrome with Evidence for Dominant-Negative Effect

PubMed Central

McEntagart, Meriel; Williamson, Kathleen A.; Rainger, Jacqueline K.; Wheeler, Ann; Seawright, Anne; De Baere, Elfride; Verdin, Hannah; Bergendahl, L. Therese; Quigley, Alan; Rainger, Joe; Dixit, Abhijit; Sarkar, Ajoy; López Laso, Eduardo; Sanchez-Carpintero, Rocio; Barrio, Jesus; Bitoun, Pierre; Prescott, Trine; Riise, Ruth; McKee, Shane; Cook, Jackie; McKie, Lisa; Ceulemans, Berten; Meire, Françoise; Temple, I. Karen; Prieur, Fabienne; Williams, Jonathan; Clouston, Penny; Németh, Andrea H.; Banka, Siddharth; Bengani, Hemant; Handley, Mark; Freyer, Elisabeth; Ross, Allyson; van Heyningen, Veronica; Marsh, Joseph A.; Elmslie, Frances; FitzPatrick, David R.

2016-01-01

Gillespie syndrome (GS) is characterized by bilateral iris hypoplasia, congenital hypotonia, non-progressive ataxia, and progressive cerebellar atrophy. Trio-based exome sequencing identified de novo mutations in ITPR1 in three unrelated individuals with GS recruited to the Deciphering Developmental Disorders study. Whole-exome or targeted sequence analysis identified plausible disease-causing ITPR1 mutations in 10/10 additional GS-affected individuals. These ultra-rare protein-altering variants affected only three residues in ITPR1: Glu2094 missense (one de novo, one co-segregating), Gly2539 missense (five de novo, one inheritance uncertain), and Lys2596 in-frame deletion (four de novo). No clinical or radiological differences were evident between individuals with different mutations. ITPR1 encodes an inositol 1,4,5-triphosphate-responsive calcium channel. The homo-tetrameric structure has been solved by cryoelectron microscopy. Using estimations of the degree of structural change induced by known recessive- and dominant-negative mutations in other disease-associated multimeric channels, we developed a generalizable computational approach to indicate the likely mutational mechanism. This analysis supports a dominant-negative mechanism for GS variants in ITPR1. In GS-derived lymphoblastoid cell lines (LCLs), the proportion of ITPR1-positive cells using immunofluorescence was significantly higher in mutant than control LCLs, consistent with an abnormality of nuclear calcium signaling feedback control. Super-resolution imaging supports the existence of an ITPR1-lined nucleoplasmic reticulum. Mice with Itpr1 heterozygous null mutations showed no major iris defects. Purkinje cells of the cerebellum appear to be the most sensitive to impaired ITPR1 function in humans. Iris hypoplasia is likely to result from either complete loss of ITPR1 activity or structure-specific disruption of multimeric interactions. PMID:27108798

Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges.

PubMed

Lakhashe, Samir K; Velu, Vijayakumar; Sciaranghella, Gaia; Siddappa, Nagadenahalli B; Dipasquale, Janet M; Hemashettar, Girish; Yoon, John K; Rasmussen, Robert A; Yang, Feng; Lee, Sandra J; Montefiori, David C; Novembre, Francis J; Villinger, François; Amara, Rama Rao; Kahn, Maria; Hu, Shiu-Lok; Li, Sufen; Li, Zhongxia; Frankel, Fred R; Robert-Guroff, Marjorie; Johnson, Welkin E; Lieberman, Judy; Ruprecht, Ruth M

2011-08-05

We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus > 90%; these RM also had strong SIV Gag-specific proliferation of CD8⁺ T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the α4β7-expressing CD4⁺ T cells; the latter have been implicated as preferential virus targets in vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Felix Hoppe-Seyler Lecture 1997. Protective antibody responses against viruses.

PubMed

Zinkernagel, R M

1997-08-01

Neutralizing antibody responses against the acute cytopathic vesicular stomatitis virus (VSV) have been studied in mice to evaluate their general characteristics including specificity, self-/non-self discrimination and memory. IgM responses are generated very early, by day 3 to 4, in a T helper cell-independent fashion and without VSV having polyclonal activating capacities. The order of the glycoprotein tips on the virus envelope (multiple, 8-10 nm distance, paracrystalline) exhibiting the neutralizing determinants are key to this prompt response. These paracrystalline identical multimeric antigens are characteristic of infectious agents and are always reacted against by B cells. Self-antigens that are accessible to B cells in the intact host are either monomeric in serum or mobile multimers on cell surfaces; these configurations need contact dependent or contact independent T help, respectively. Because T help is tolerant against self-antigens, no anti-self B cell responses are usually induced against monomeric self-antigens. If collagen or DNA (rigid multimeric self-antigens) become accessible, however, they may become targets of auto-antibody responses. The antibody repertoire against VSV is partially contained in the germline and partially is generated by somatic mutation; they seem not to undergo affinity-maturation. In any case protection against lethal infection is dependent upon strictly T helper cell dependent IgG generated by day 6 to 7 and reaches a protective level of about 1-10 micrograms/ml. Interesting affinity/avidity and onrate above a minimal threshold are of no apparent advantage for protection in vivo. Maintenance of these antibody levels by antigen depots, and not the presence of memory B cells alone, is key to providing protective immunological memory. Collectively these data suggest that studying biologically important protective antibody responses may modify some of the parameters that have been defined by studying hapten specific antibody responses.

Enabling Efficient and Confident Annotation of LC−MS Metabolomics Data through MS1 Spectrum and Time Prediction

DOE PAGES

Broeckling, Corey D.; Ganna, Andrea; Layer, Mark; ...

2016-09-08

Liquid chromatography coupled to electrospray ionization-mass spectrometry (LC–ESI-MS) is a versatile and robust platform for metabolomic analysis. However, while ESI is a soft ionization technique, in-source phenomena including multimerization, nonproton cation adduction, and in-source fragmentation complicate interpretation of MS data. Here, we report chromatographic and mass spectrometric behavior of 904 authentic standards collected under conditions identical to a typical nontargeted profiling experiment. The data illustrate that the often high level of complexity in MS spectra is likely to result in misinterpretation during the annotation phase of the experiment and a large overestimation of the number of compounds detected. However, ourmore » analysis of this MS spectral library data indicates that in-source phenomena are not random but depend at least in part on chemical structure. These nonrandom patterns enabled predictions to be made as to which in-source signals are likely to be observed for a given compound. Using the authentic standard spectra as a training set, we modeled the in-source phenomena for all compounds in the Human Metabolome Database to generate a theoretical in-source spectrum and retention time library. A novel spectral similarity matching platform was developed to facilitate efficient spectral searching for nontargeted profiling applications. Taken together, this collection of experimental spectral data, predictive modeling, and informatic tools enables more efficient, reliable, and transparent metabolite annotation.« less

Evaluation of three simple direct or indirect carbonyl detection methods for characterization of oxidative modifications of proteins.

PubMed

Vásquez-Garzón, Verónica R; Rouimi, Patrick; Jouanin, Isabelle; Waeg, Georg; Zarkovic, Neven; Villa-Treviño, Saul; Guéraud, Françoise

2012-05-01

Among disruptions induced by oxidative stress, modifications of proteins, particularly irreversible carbonylation, are associated with the development of several diseases, including cardiovascular diseases, neurodegenerative diseases, and cancer. Carbonylation of proteins can occur directly or indirectly through the adduction of lipid oxidation products. In this study, three classical and easy-to-perform techniques to detect direct or indirect carbonylation of proteins were compared. A model protein apomyoglobin and a complex mixture of rat liver cytosolic proteins were exposed to cumene hydroperoxide oxidation or adduction to the lipid peroxidation product 4-hydroxynonenal in order to test direct or indirect carbonylation, respectively. The technique using a specific anti-4-hydroxynonenal-histidine adduct antibody was effective to detect in vitro modification of model apomyoglobin and cytosolic proteins by 4-hydroxynonenal but not by direct carbonylation which was achieved by techniques using biotin-coupled hydrazide or dinitrophenylhydrazine derivatization of carbonyls. Sequential use of these methods enabled the detection of both direct and indirect carbonyl modification in proteins, although constitutively biotinylated proteins were detected by biotin-hydrazide. Although rather classical and efficient, methods for carbonyl detection on proteins in oxidative stress studies may be biased by some artifactual detections and complicated by proteins multimerizations. The use of more and more specific available antibodies is recommended to complete detection of lipid peroxidation product adducts on proteins.

Enabling Efficient and Confident Annotation of LC-MS Metabolomics Data through MS1 Spectrum and Time Prediction

DOE PAGES

Broeckling, Corey D.; Ganna, Andrea; Layer, Mark; ...

2016-08-25

Liquid chromatography coupled to electrospray ionization-mass spectrometry (LC–ESI-MS) is a versatile and robust platform for metabolomic analysis. However, while ESI is a soft ionization technique, in-source phenomena including multimerization, nonproton cation adduction, and in-source fragmentation complicate interpretation of MS data. Here, we report chromatographic and mass spectrometric behavior of 904 authentic standards collected under conditions identical to a typical nontargeted profiling experiment. The data illustrate that the often high level of complexity in MS spectra is likely to result in misinterpretation during the annotation phase of the experiment and a large overestimation of the number of compounds detected. However, ourmore » analysis of this MS spectral library data indicates that in-source phenomena are not random but depend at least in part on chemical structure. These nonrandom patterns enabled predictions to be made as to which in-source signals are likely to be observed for a given compound. Using the authentic standard spectra as a training set, we modeled the in-source phenomena for all compounds in the Human Metabolome Database to generate a theoretical in-source spectrum and retention time library. A novel spectral similarity matching platform was developed to facilitate efficient spectral searching for nontargeted profiling applications. Taken together, this collection of experimental spectral data, predictive modeling, and informatic tools enables more efficient, reliable, and transparent metabolite annotation.« less

Identification of six novel mutations in Iranian patients with maple syrup urine disease and their in silico analysis.

PubMed

Abiri, Maryam; Karamzadeh, Razieh; Karimipoor, Morteza; Ghadami, Shirin; Alaei, Mohammad Reza; Bagheri, Samira Dabagh; Bagherian, Hamideh; Setoodeh, Aria; Noori-Daloii, Mohammad Reza; Sirous Zeinali

2016-04-01

Maple syrup urine disease (MSUD) is a rare inborn error of branched-chain amino acid metabolism. The disease prevalence is higher in populations with elevated rate of consanguineous marriages such as Iran. Different types of disease causing mutations have been previously reported in BCKDHA, BCKDHB, DBT and DLD genes known to be responsible for MSUD phenotype. In this study, two sets of multiplex polymorphic STR (Short Tandem Repeat) markers linked to the above genes were used to aid in homozygosity mapping in order to find probable pathogenic change(s) in the studied families. The families who showed homozygote haplotype for the BCKDHA gene were subsequently sequenced. Our findings showed that exons 2, 4 and 6 contain most of the mutations which are novel. The changes include two single nucleotide deletion (i.e. c. 143delT and c.702delT), one gross deletion covering the whole exon four c.(375+1_376-1)_(8849+1_885-1), two splice site changes (c.1167+1G>T, c. 288+1G>A), and one point mutation (c.731G>A). Computational approaches were used to analyze these two novel mutations in terms of their impact on protein structure. Computational structural modeling indicated that these mutations might affect structural stability and multimeric assembly of branched-chain α-keto acid dehydrogenase complex (BCKDC). Copyright © 2016. Published by Elsevier B.V.

Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix

DOE PAGES

Horton, J. R.; Wang, H.; Mabuchi, M. Y.; ...

2014-09-27

MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNAmore » molecules bound specifically to the tetramer and engaged with the enzyme's two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act.« less

Monomer-dimer control of the ColE1 P(cer) promoter.

PubMed

Chatwin, H M; Summers, D K

2001-11-01

XerCD-mediated recombination at cer converts multimers of plasmid ColE1 to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss. In addition to XerCD, recombination requires the accessory factors ArgR and PepA. The promoter P(cer), located centrally within cer, is also required for stable plasmid maintenance. P(cer) is active in plasmid multimers and directs transcription of a short RNA, Rcd, which appears to inhibit cell division. It has been proposed that Rcd is part of a checkpoint which ensures that multimer resolution is complete before the cell divides. This study has shown that ArgR does not act as a transcriptional repressor of P(cer) in plasmid monomers. P(cer) is unusual in that the -35 and -10 hexamers are separated by only 15 bp and this study has demonstrated that increasing this to a more conventional spacing results in elevated activity. An increase to 17 bp resulted in a 10- to 20-fold increase in activity, while smaller effects were seen when the spacer was increased to 16 bp or 18 bp. These observations are consistent with the hypothesis that P(cer) activation involves realignment of the -35 and -10 sequences within a recombinational synaptic complex. This predicts that a 17 bp spacer promoter derivative should be down-regulated by plasmid multimerization, and this is confirmed experimentally.

Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase.

PubMed

Stepper, Peter; Kungulovski, Goran; Jurkowska, Renata Z; Chandra, Tamir; Krueger, Felix; Reinhardt, Richard; Reik, Wolf; Jeltsch, Albert; Jurkowski, Tomasz P

2017-02-28

DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a-Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20-30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The HIV-1 integrase-LEDGF allosteric inhibitor MUT-A: resistance profile, impairment of virus maturation and infectivity but without influence on RNA packaging or virus immunoreactivity.

PubMed

Amadori, Céline; van der Velden, Yme Ubeles; Bonnard, Damien; Orlov, Igor; van Bel, Nikki; Le Rouzic, Erwann; Miralles, Laia; Brias, Julie; Chevreuil, Francis; Spehner, Daniele; Chasset, Sophie; Ledoussal, Benoit; Mayr, Luzia; Moreau, François; García, Felipe; Gatell, José; Zamborlini, Alessia; Emiliani, Stéphane; Ruff, Marc; Klaholz, Bruno P; Moog, Christiane; Berkhout, Ben; Plana, Montserrat; Benarous, Richard

2017-11-09

HIV-1 Integrase (IN) interacts with the cellular co-factor LEDGF/p75 and tethers the HIV preintegration complex to the host genome enabling integration. Recently a new class of IN inhibitors was described, the IN-LEDGF allosteric inhibitors (INLAIs). Designed to interfere with the IN-LEDGF interaction during integration, the major impact of these inhibitors was surprisingly found on virus maturation, causing a reverse transcription defect in target cells. Here we describe the MUT-A compound as a genuine INLAI with an original chemical structure based on a new type of scaffold, a thiophene ring. MUT-A has all characteristics of INLAI compounds such as inhibition of IN-LEDGF/p75 interaction, IN multimerization, dual antiretroviral (ARV) activities, normal packaging of genomic viral RNA and complete Gag protein maturation. MUT-A has more potent ARV activity compared to other INLAIs previously reported, but similar profile of resistance mutations and absence of ARV activity on SIV. HIV-1 virions produced in the presence of MUT-A were non-infectious with the formation of eccentric condensates outside of the core. In studying the immunoreactivity of these non-infectious virions, we found that inactivated HIV-1 particles were captured by anti-HIV-specific neutralizing and non-neutralizing antibodies (b12, 2G12, PGT121, 4D4, 10-1074, 10E8, VRC01) with efficiencies comparable to non-treated virus. Autologous CD4 + T lymphocyte proliferation and cytokine induction by monocyte-derived dendritic cells (MDDC) pulsed either with MUT-A-inactivated HIV or non-treated HIV were also comparable. Although strongly defective in infectivity, HIV-1 virions produced in the presence of the MUT-A INLAI have a normal protein and genomic RNA content as well as B and T cell immunoreactivities comparable to non-treated HIV-1. These inactivated viruses might form an attractive new approach in vaccine research in an attempt to study if this new type of immunogen could elicit an immune response against HIV-1 in animal models.

Identification of beta-2 as a key cell adhesion molecule in PCa cell neurotropic behavior: a novel ex vivo and biophysical approach.

PubMed

Jansson, Keith H; Castillo, Deborah G; Morris, Joseph W; Boggs, Mary E; Czymmek, Kirk J; Adams, Elizabeth L; Schramm, Lawrence P; Sikes, Robert A

2014-01-01

Prostate cancer (PCa) is believed to metastasize through the blood/lymphatics systems; however, PCa may utilize the extensive innervation of the prostate for glandular egress. The interaction of PCa and its nerve fibers is observed in 80% of PCa and is termed perineural invasion (PNI). PCa cells have been observed traveling through the endoneurium of nerves, although the underlying mechanisms have not been elucidated. Voltage sensitive sodium channels (VSSC) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two auxiliary beta (β) subunits with inherent cell adhesion molecule (CAM) functions. The beta-2 isoform (gene SCN2B) interacts with several neural CAMs, while interacting putatively with other prominent neural CAMs. Furthermore, beta-2 exhibits elevated mRNA and protein levels in highly metastatic and castrate-resistant PCa. When overexpressed in weakly aggressive LNCaP cells (2BECFP), beta-2 alters LNCaP cell morphology and enhances LNCaP cell metastasis associated behavior in vitro. We hypothesize that PCa cells use beta-2 as a CAM during PNI and subsequent PCa metastasis. The objective of this study was to determine the effect of beta-2 expression on PCa cell neurotropic metastasis associated behavior. We overexpressed beta-2 as a fusion protein with enhanced cyan fluorescence protein (ECFP) in weakly aggressive LNCaP cells and observed neurotropic effects utilizing our novel ex vivo organotypic spinal cord co-culture model, and performed functional assays with neural matrices and atomic force microscopy. With increased beta-2 expression, PCa cells display a trend of enhanced association with nerve axons. On laminin, a neural CAM, overexpression of beta-2 enhances PCa cell migration, invasion, and growth. 2BECFP cells exhibit marked binding affinity to laminin relative to LNECFP controls, and recombinant beta-2 ectodomain elicits more binding events to laminin than BSA control. Functional overexpression of VSSC beta subunits in PCa may mediate PCa metastatic behavior through association with neural matrices.

Generation of porcine reproductive and respiratory syndrome (PRRS) virus-like-particles (VLPs) with different protein composition.

PubMed

García Durán, Marga; Costa, Sofia; Sarraseca, Javier; de la Roja, Nuria; García, Julia; García, Isabel; Rodríguez, Maria José

2016-10-01

The causative agent of Porcine Reproductive and Respiratory Syndrome (PRRS) is an enveloped ssRNA (+) virus belonging to the Arteriviridae family. Gp5 and M proteins form disulfide-linked heterodimers that constitute the major components of PRRSV envelope. Gp2, Gp3, Gp4 and E are the minor structural proteins, being the first three incorporated as multimeric complexes in the virus surface. The disease has become one of the most important causes of economic losses in the swine industry. Despite efforts to design an effective vaccine, the available ones allow only partial protection. In the last years, VLPs have become good vaccine alternatives because of safety issues and their potential to activate both branches of the immunological response. The characteristics of recombinant baculoviruses as heterologous expression system have been exploited for the production of VLPs of a wide variety of viruses. In this work, two multiple baculovirus expression vectors (BEVs) with PRRS virus envelope proteins were engineered in order to generate PRRS VLPs: on the one hand, Gp5 and M cDNAs were cloned to generate the pBAC-Gp5M vector; on the other hand, Gp2, Gp3, Gp4 and E cDNAs have been cloned to generate the pBAC-Gp234E vector. The corresponding recombinant baculoviruses BAC-Gp5M and BAC-Gp234E were employed to produce two types of VLPs: basic Gp5M VLPs, by the simultaneous expression of Gp5 and M proteins; and complete VLPs, by the co-expression of the six PRRS proteins after co-infection. The characterization of VLPs by Western blot confirmed the presence of the recombinant proteins using the available specific antibodies (Abs). The analysis by Electron microscopy showed that the two types of VLPs were indistinguishable between them, being similar in shape and size to the native PRRS virus. This system represents a potential alternative for vaccine development and a useful tool to study the implication of specific PRRS proteins in the response against the virus. Copyright © 2016 Elsevier B.V. All rights reserved.

BALBES: a molecular-replacement pipeline.

PubMed

Long, Fei; Vagin, Alexei A; Young, Paul; Murshudov, Garib N

2008-01-01

The number of macromolecular structures solved and deposited in the Protein Data Bank (PDB) is higher than 40 000. Using this information in macromolecular crystallography (MX) should in principle increase the efficiency of MX structure solution. This paper describes a molecular-replacement pipeline, BALBES, that makes extensive use of this repository. It uses a reorganized database taken from the PDB with multimeric as well as domain organization. A system manager written in Python controls the workflow of the process. Testing the current version of the pipeline using entries from the PDB has shown that this approach has huge potential and that around 75% of structures can be solved automatically without user intervention.

Purification, isolation, crystallization, and preliminary X-ray diffraction study of the BTB domain of the centrosomal protein 190 from Drosophila melanogaster

NASA Astrophysics Data System (ADS)

Boyko, K. M.; Nikolaeva, A. Yu.; Kachalova, G. S.; Bonchuk, A. N.; Popov, V. O.

2017-11-01

The spatial organization of the genome is controlled by a special class of architectural proteins, including proteins containing BTB domains that are able to dimerize or multimerize. The centrosomal protein 190 is one of such architectural proteins. The purification, crystallization, and preliminary X-ray diffraction study of the BTB domain of the centrosomal protein 190 are reported. The crystallization conditions were found by the vapor-diffusion technique. The crystals diffracted to 1.5 Å resolution and belonged to sp. gr. P3221. The structure was solved by the molecular replacement method. The structure refinement is currently underway.

RNA Nanotechnology: Engineering, Assembly and Applications in Detection, Gene Delivery and Therapy

PubMed Central

Guo, Peixuan

2010-01-01

Biological macromolecules including DNA, RNA, and proteins, have intrinsic features that make them potential building blocks for the bottom-up fabrication of nanodevices. RNA is unique in nanoscale fabrication due to its amazing diversity of function and structure. RNA molecules can be designed and manipulated with a level of simplicity characteristic of DNA while possessing versatility in structure and function similar to that of proteins. RNA molecules typically contain a large variety of single stranded loops suitable for inter- and intra-molecular interaction. These loops can serve as mounting dovetails obviating the need for external linking dowels in fabrication and assembly. The self-assembly of nanoparticles from RNA involves cooperative interaction of individual RNA molecules that spontaneously assemble in a predefined manner to form a larger two- or three-dimensional structure. Within the realm of self-assembly there are two main categories, namely template and non-template. Template assembly involves interaction of RNA molecules under the influence of specific external sequence, forces, or spatial constraints such as RNA transcription, hybridization, replication, annealing, molding, or replicas. In contrast, non-template assembly involves formation of a larger structure by individual components without the influence of external forces. Examples of non-template assembly are ligation, chemical conjugation, covalent linkage, and loop/loop interaction of RNA, especially the formation of RNA multimeric complexes. The best characterized RNA multiplier and the first to be described in RNA nanotechnological application is the motor pRNA of bacteriophage phi29 which form dimers, trimers, and hexamers, via hand-in-hand interaction. phi29 pRNA can be redesigned to form a variety of structures and shapes including twins, tetramers, rods, triangles, and 3D arrays several microns in size via interaction of programmed helical regions and loops. 3D RNA array formation requires a defined nucleotide number for twisting and a palindromic sequence. Such arrays are unusually stable and resistant to a wide range of temperatures, salt concentrations, and pH. Both the therapeutic siRNA or ribozyme and a receptor-binding RNA aptamer or other ligands have been engineered into individual pRNAs. Individual chimeric RNA building blocks harboring siRNA or other therapeutic molecules have been fabricated subsequently into a trimer through hand-in-hand interaction of the engineered right and left interlocking RNA loops. The incubation of these particles containing the receptor-binding aptamer or other ligands results in the binding and co-entry of trivalent therapeutic particles into cells. Such particles were subsequently shown to modulate the apoptosis of cancer cells in both cell cultures and animal trials. The use of such antigen-free 20–40 nm particles holds promise for the repeated long-term treatment of chronic diseases. Other potentially useful RNA molecules that form multimers include HIV RNA that contain kissing loop to form dimers, tecto-RNA that forms a “jigsaw puzzle,” and the Drosophila bicoid mRNA that forms multimers via “hand-by-arm” interactions. Applications of RNA molecules involving replication, molding, embossing, and other related techniques, have recently been described that allow the utilization of a variety of materials to enhance diversity and resolution of nanomaterials. It should eventually be possible to adapt RNA to facilitate construction of ordered, patterned, or pre-programmed arrays or superstructures. Given the potential for 3D fabrication, the chance to produce reversible self-assembly, and the ability of self-repair, editing and replication, RNA self-assembly will play an increasingly significant role in integrated biological nanofabrication. A random 100-nucleotide RNA library may exist in 1.6 × 1060 varieties with multifarious structure to serve as a vital system for efficient fabrication, with a complexity and diversity far exceeding that of any current nanoscale system. This review covers the basic concepts of RNA structure and function, certain methods for the study of RNA structure, the approaches for engineering or fabricating RNA into nanoparticles or arrays, and special features of RNA molecules that form multimers. The most recent development in exploration of RNA nanoparticles for pathogen detection, drug/gene delivery, and therapeutic application is also introduced in this review. PMID:16430131

Novel Pentameric Structure of the Diarrhea-Inducing Region of the Rotavirus Enterotoxigenic Protein NSP4▿

PubMed Central

Chacko, Anita R.; Arifullah, Mohammed; Sastri, Narayan P.; Jeyakanthan, Jeyaraman; Ueno, Go; Sekar, Kanagaraj; Read, Randy J.; Dodson, Eleanor J.; Rao, Durga C.; Suguna, Kaza

2011-01-01

A novel pentameric structure which differs from the previously reported tetrameric form of the diarrhea-inducing region of the rotavirus enterotoxin NSP4 is reported here. A significant feature of this pentameric form is the absence of the calcium ion located in the core region of the tetrameric structures. The lysis of cells, the crystallization of the region spanning residues 95 to 146 of NSP4 (NSP495-146) of strain ST3 (ST3:NSP495-146) at acidic pH, and comparative studies of the recombinant purified peptide under different conditions by size-exclusion chromatography (SEC) and of the crystal structures suggested pH-, Ca2+-, and protein concentration-dependent oligomeric transitions in the peptide. Since the NSP495-146 mutant lacks the N-terminal amphipathic domain (AD) and most of the C-terminal flexible region (FR), to demonstrate that the pentameric transition is not a consequence of the lack of the N- and C-terminal regions, glutaraldehyde cross-linking of the ΔN72 and ΔN94 mutant proteins, which contain or lack the AD, respectively, but possess the complete C-terminal FR, was carried out. The results indicate the presence of pentamers in preparations of these longer mutants. Detailed SEC analyses of ΔN94 prepared under different conditions, however, revealed protein concentration-dependent but metal ion- and pH-independent pentamer accumulation at high concentrations which dissociated into tetramers and lower oligomers at low protein concentrations. While calcium appeared to stabilize the tetramer, magnesium in particular stabilized the dimer. ΔN72 existed primarily in the multimeric form under all conditions. These findings of a calcium-free NSP4 pentamer and its concentration-dependent and largely calcium-independent oligomeric transitions open up a new dimension in an understanding of the structural basis of its multitude of functions. PMID:21917949

Involvement of PlsX and the acyl-phosphate dependent sn-glycerol-3-phosphate acyltransferase PlsY in the initial stage of glycerolipid synthesis in Bacillus subtilis.

PubMed

Hara, Yoshinori; Seki, Masahide; Matsuoka, Satoshi; Hara, Hiroshi; Yamashita, Atsushi; Matsumoto, Kouji

2008-12-01

The gene responsible for the first acylation of sn-glycerol-3-phosphate (G3P) in Bacillus subtilis has not yet been determined with certainty. The product of this first acylation, lysophosphatidic acid (LPA), is subsequently acylated again to form phosphatidic acid (PA), the primary precursor to membrane glycerolipids. A novel G3P acyltransferase (GPAT), the gene product of plsY, which uses acyl-phosphate formed by the plsX gene product, has recently been found to synthesize LPA in Streptococcus pneumoniae. We found that in B. subtilis growth arrests after repression of either a plsY homologue or a plsX homologue were overcome by expression of E. coli plsB, which encodes an acyl-acylcarrier protein (acyl-ACP)-dependent GPAT, although in the case of plsX repression a high level of plsB expression was required. B. subtilis has, therefore, a capability to use the acyl-ACP dependent GPAT of PlsB. Simultaneous expression of plsY and plsX suppressed the glycerol requirement of a strict glycerol auxotrophic derivative of the E. coli plsB26 mutant, although either one alone did not. Membrane fractions from B. subtilis cells catalyzed palmitoylphosphate-dependent acylation of [14C]-labeled G3P to synthesize [14C]-labeled LPA, whereas those from DeltaplsY cells did not. The results indicate unequivocally that PlsY is an acyl-phosphate dependent GPAT. Expression of plsX corrected the glycerol auxotrophy of a DeltaygiH (the deleted allele of an E. coli homologue of plsY) derivative of BB26-36 (plsB26 plsX50), suggesting an essential role of plsX other than substrate supply for acyl-phosphate dependent LPA synthesis. Two-hybrid examinations suggested that PlsY is associated with PlsX and that each may exist in multimeric form.

Cooperative DNA binding and protein/DNA fiber formation increases the activity of the Dnmt3a DNA methyltransferase.

PubMed

Emperle, Max; Rajavelu, Arumugam; Reinhardt, Richard; Jurkowska, Renata Z; Jeltsch, Albert

2014-10-24

The Dnmt3a DNA methyltransferase has been shown to bind cooperatively to DNA and to form large multimeric protein/DNA fibers. However, it has also been reported to methylate DNA in a processive manner, a property that is incompatible with protein/DNA fiber formation. We show here that the DNA methylation rate of Dnmt3a increases more than linearly with increasing enzyme concentration on a long DNA substrate, but not on a short 30-mer oligonucleotide substrate. We also show that addition of a catalytically inactive Dnmt3a mutant, which carries an amino acid exchange in the catalytic center, increases the DNA methylation rate by wild type Dnmt3a on the long substrate but not on the short one. In agreement with this finding, preincubation experiments indicate that stable protein/DNA fibers are formed on the long, but not on the short substrate. In addition, methylation experiments with substrates containing one or two CpG sites did not provide evidence for a processive mechanism over a wide range of enzyme concentrations. These data clearly indicate that Dnmt3a binds to DNA in a cooperative reaction and that the formation of stable protein/DNA fibers increases the DNA methylation rate. Fiber formation occurs at low μm concentrations of Dnmt3a, which are in the range of Dnmt3a concentrations in the nucleus of embryonic stem cells. Understanding the mechanism of Dnmt3a is of vital importance because Dnmt3a is a hotspot of somatic cancer mutations one of which has been implicated in changing Dnmt3a processivity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A switch from parallel to antiparallel strand orientation in a coiled-coil X-ray structure via two core hydrophobic mutations

DOE PAGES

Malashkevich, Vladimir N.; Higgins, Chelsea D.; Almo, Steven C.; ...

2015-05-06

The coiled-coil is one of the most ubiquitous and well studied protein structural motifs. Significant effort has been devoted to dissecting subtle variations of the typical heptad repeat sequence pattern that can designate larger topological features such as relative α-helical orientation and oligomer size. Here in this paper we report the X-ray structure of a model coiled-coil peptide, HA2-Del-L2seM, which forms an unanticipated core antiparallel dimer with potential sites for discrete higher-order multimerization (trimer or tetramer). In the X-ray structure, a third, partially-ordered α-helix is weakly associated with the antiparallel dimer and analytical ultracentrifugation experiments indicate the peptide forms amore » well-defined tetramer in solution. The HA2-Del-L2seM sequence is closely related to a parent model peptide, HA2-Del, which we previously reported adopts a parallel trimer; HA2-Del-L2seM differs by only hydrophobic leucine to selenomethione mutations and thus this subtle difference is sufficient to switch both relative α-helical topology and number of α-helices participating in the coiled-coil. Comparison of the X-ray structures of HA2-Del-L2seM (reported here) with the HA2-Del parent (reported previously) reveals novel interactions involving the selenomethionine residues that promote antiparallel coiled-coil configuration and preclude parallel trimer formation. Finally, these novel atomic insights are instructive for understanding subtle features that can affect coiled-coil topology and provide additional information for design of antiparallel coiled-coils.« less

Surface Localization of Zein Storage Proteins in Starch Granules from Maize Endosperm1

PubMed Central

Mu-Forster, Chen; Wasserman, Bruce P.

1998-01-01

Starch granules from maize (Zea mays) contain a characteristic group of polypeptides that are tightly associated with the starch matrix (C. Mu-Forster, R. Huang, J.R. Powers, R.W. Harriman, M. Knight, G.W. Singletary, P.L. Keeling, B.P. Wasserman [1996] Plant Physiol 111: 821–829). Zeins comprise about 50% of the granule-associated proteins, and in this study their spatial distribution within the starch granule was determined. Proteolysis of starch granules at subgelatinization temperatures using the thermophilic protease thermolysin led to selective removal of the zeins, whereas granule-associated proteins of 32 kD or above, including the waxy protein, starch synthase I, and starch-branching enzyme IIb, remained refractory to proteolysis. Granule-associated proteins from maize are therefore composed of two distinct classes, the surface-localized zeins of 10 to 27 kD and the granule-intrinsic proteins of 32 kD or higher. The origin of surface-localized δ-zein was probed by comparing δ-zein levels of starch granules obtained from homogenized whole endosperm with granules isolated from amyloplasts. Starch granules from amyloplasts contained markedly lower levels of δ-zein relative to granules prepared from whole endosperm, thus indicating that δ-zein adheres to granule surfaces after disruption of the amyloplast envelope. Cross-linking experiments show that the zeins are deposited on the granule surface as aggregates. In contrast, the granule-intrinsic proteins are prone to covalent modification, but do not form intermolecular cross-links. We conclude that individual granule intrinsic proteins exist as monomers and are not deposited in the form of multimeric clusters within the starch matrix. PMID:9536075

The Replacement of 10 Non-Conserved Residues in the Core Protein of JFH-1 Hepatitis C Virus Improves Its Assembly and Secretion

PubMed Central

Etienne, Loïc; Blanchard, Emmanuelle; Boyer, Audrey; Desvignes, Virginie; Gaillard, Julien; Meunier, Jean-Christophe; Roingeard, Philippe; Hourioux, Christophe

2015-01-01

Hepatitis C virus (HCV) assembly is still poorly understood. It is thought that trafficking of the HCV core protein to the lipid droplet (LD) surface is essential for its multimerization and association with newly synthesized HCV RNA to form the viral nucleocapsid. We carried out a mapping analysis of several complete HCV genomes of all genotypes, and found that the genotype 2 JFH-1 core protein contained 10 residues different from those of other genotypes. The replacement of these 10 residues of the JFH-1 strain sequence with the most conserved residues deduced from sequence alignments greatly increased virus production. Confocal microscopy of the modified JFH-1 strain in cell culture showed that the mutated JFH-1 core protein, C10M, was present mostly at the endoplasmic reticulum (ER) membrane, but not at the surface of the LDs, even though its trafficking to these organelles was possible. The non-structural 5A protein of HCV was also redirected to ER membranes and colocalized with the C10M core protein. Using a Semliki forest virus vector to overproduce core protein, we demonstrated that the C10M core protein was able to form HCV-like particles, unlike the native JFH-1 core protein. Thus, the substitution of a few selected residues in the JFH-1 core protein modified the subcellular distribution and assembly properties of the protein. These findings suggest that the early steps of HCV assembly occur at the ER membrane rather than at the LD surface. The C10M-JFH-1 strain will be a valuable tool for further studies of HCV morphogenesis. PMID:26339783

DNA double-strand break in vivo at the 3' extremity of exons located upstream of group II introns. Senescence and circular DNA introns in Podospora mitochondria.

PubMed

Sainsard-Chanet, A; Begel, O; Belcour, L

1994-10-07

In the filamentous fungus Podospora anserina, the unavoidable phenomenon of senescence is associated with the amplification of the first intron of the mitochondrial cox1 that accumulates as circular DNA molecules consisting of tandem repeats. This group II intron (cox1-i1 or alpha) is able to transpose and contains an open reading frame with significant amino acid similarity with reverse transcriptases. The generation of these intronic circular DNA molecules, their amplification and their involvement in the senescence process are unresolved questions. We demonstrate here that: (1) another group II intron, the fourth intron of gene cox1, cox1-i4, is also able to give precise DNA end to end junctions; (2) this intronic sequence can be found amplified during senescence, although to a lesser extent than cox1-i1; (3) the amplification of the DNA multimeric cox1-i1 molecules likely does not proceed by autonomous replication; (4) the generation of the DNA intronic circles does not require efficient intron splicing; (5) a DNA double-strand break occurs in vivo at the 3' extremity of the cox1-e1 and cox1-e4 exons preceding the group II introns that form circular DNAs. On the whole, these results show that the ability to form DNA circular molecules is a property of some group II introns and they demonstrate the occurrence of a specific DNA cleavage at or near the integration site of these group II introns. The results strongly suggest that this cleavage is involved in the formation of the group II intronic DNA circles and could also be involved in the phenomenon of group II intron homing.

Subunit Stabilization and Polyethylene Glycolation of Cocaine Esterase Improves In Vivo Residence Time

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

Narasimhan, Diwahar; Collins, Gregory T.; Nance, Mark R.

No small-molecule therapeutic is available to treat cocaine addiction, but enzyme-based therapy to accelerate cocaine hydrolysis in serum has gained momentum. Bacterial cocaine esterase (CocE) is the fastest known native enzyme that hydrolyzes cocaine. However, its lability at 37 C has limited its therapeutic potential. Cross-linking subunits through disulfide bridging is commonly used to stabilize multimeric enzymes. Herein we use structural methods to guide the introduction of two cysteine residues within dimer interface of CocE to facilitate intermolecular disulfide bond formation. The disulfide-crosslinked enzyme displays improved thermostability, particularly when combined with previously described mutations that enhance stability (T172R-G173Q). The newlymore » modified enzyme yielded an extremely stable form of CocE (CCRQ-CocE) that retained greater than 90% of its activity after 41 days at 37 C, representing an improvement of more than 4700-fold over the wild-type enzyme. CCRQ-CocE could also be modified by polyethylene glycol (PEG) polymers, which improved its in vivo residence time from 24 to 72 h, as measured by a cocaine lethality assay, by self-administration in rodents, and by measurement of inhibition of cocaine-induced cardiovascular effects in rhesus monkeys. PEG-CCRQ elicited negligible immune response in rodents. Subunit stabilization and PEGylation has thus produced a potential protein therapeutic with markedly higher stability both in vitro and in vivo.« less

Expression of cholinesterase gene(s) in human brain tissues: translational evidence for multiple mRNA species.

PubMed Central

Soreq, H; Zevin-Sonkin, D; Razon, N

1984-01-01

To resolve the origin(s) of the molecular heterogeneity of human nervous system cholinesterases (ChEs), we used Xenopus oocytes, which produce biologically active ChE when microinjected with unfractionated brain mRNA. The RNA was prepared from primary gliomas, meningiomas and embryonic brain, each of which expresses ChE activity with distinct substrate specificities and molecular forms. Sucrose gradient fractionation of DMSO-denatured mRNA from these sources revealed three size classes of ChE-inducing mRNAs, sedimenting at approximately 32S, 20S and 9S. The amounts of these different classes of ChE-inducing mRNAs varied between the three tissue sources examined. To distinguish between ChEs produced in oocytes and having different substrate specificities, their activity was determined in the presence of selective inhibitors. Both 'true' (acetylcholine hydrolase, EC 3.1.1.7) and 'pseudo' (acylcholine acylhydrolase, EC 3.1.1.8) multimeric cholinesterase activities were found in the mRNA-injected oocytes. Moreover, human brain mRNAs inducing 'true' and 'pseudo' ChE activities had different size distribution, indicating that different mRNAs might be translated into various types of ChEs. These findings imply that the heterogeneity of ChEs in the human nervous system is not limited to the post-translational level, but extends to the level of mRNA. PMID:6745236

von Willebrand factor, Jedi knight of the bloodstream.

PubMed

Springer, Timothy A

2014-08-28

When blood vessels are cut, the forces in the bloodstream increase and change character. The dark side of these forces causes hemorrhage and death. However, von Willebrand factor (VWF), with help from our circulatory system and platelets, harnesses the same forces to form a hemostatic plug. Force and VWF function are so closely intertwined that, like members of the Jedi Order in the movie Star Wars who learn to use "the Force" to do good, VWF may be considered the Jedi knight of the bloodstream. The long length of VWF enables responsiveness to flow. The shape of VWF is predicted to alter from irregularly coiled to extended thread-like in the transition from shear to elongational flow at sites of hemostasis and thrombosis. Elongational force propagated through the length of VWF in its thread-like shape exposes its monomers for multimeric binding to platelets and subendothelium and likely also increases affinity of the A1 domain for platelets. Specialized domains concatenate and compact VWF during biosynthesis. A2 domain unfolding by hydrodynamic force enables postsecretion regulation of VWF length. Mutations in VWF in von Willebrand disease contribute to and are illuminated by VWF biology. I attempt to integrate classic studies on the physiology of hemostatic plug formation into modern molecular understanding, and point out what remains to be learned. © 2014 by The American Society of Hematology.

Subunit Stabilization and Polyethylene Glycolation of Cocaine Esterase Improves In Vivo Residence TimeS⃞

PubMed Central

Narasimhan, Diwahar; Collins, Gregory T.; Nance, Mark R.; Nichols, Joseph; Edwald, Elin; Chan, Jimmy; Ko, Mei-Chuan; Woods, James H.; Tesmer, John J. G.

2011-01-01

No small-molecule therapeutic is available to treat cocaine addiction, but enzyme-based therapy to accelerate cocaine hydrolysis in serum has gained momentum. Bacterial cocaine esterase (CocE) is the fastest known native enzyme that hydrolyzes cocaine. However, its lability at 37°C has limited its therapeutic potential. Cross-linking subunits through disulfide bridging is commonly used to stabilize multimeric enzymes. Herein we use structural methods to guide the introduction of two cysteine residues within dimer interface of CocE to facilitate intermolecular disulfide bond formation. The disulfide-crosslinked enzyme displays improved thermostability, particularly when combined with previously described mutations that enhance stability (T172R-G173Q). The newly modified enzyme yielded an extremely stable form of CocE (CCRQ-CocE) that retained greater than 90% of its activity after 41 days at 37°C, representing an improvement of more than 4700-fold over the wild-type enzyme. CCRQ-CocE could also be modified by polyethylene glycol (PEG) polymers, which improved its in vivo residence time from 24 to 72 h, as measured by a cocaine lethality assay, by self-administration in rodents, and by measurement of inhibition of cocaine-induced cardiovascular effects in rhesus monkeys. PEG-CCRQ elicited negligible immune response in rodents. Subunit stabilization and PEGylation has thus produced a potential protein therapeutic with markedly higher stability both in vitro and in vivo. PMID:21890748

New features of mitochondrial DNA replication system in yeast and man.

PubMed

Lecrenier, N; Foury, F

2000-04-04

In this review, we sum up the research carried out over two decades on mitochondrial DNA (mtDNA) replication, primarily by comparing this system in Saccharomyces cerevisiae and Homo sapiens. Brief incursions into systems of other organisms have also been achieved when they provide new information.S. cerevisiae and H. sapiens mitochondrial DNA (mtDNA) have been thought for a long time to share closely related architecture and replication mechanisms. However, recent studies suggest that mitochondrial genome of S. cerevisiae may be formed, at least partially, from linear multimeric molecules, while human mtDNA is circular. Although several proteins involved in the replication of these two genomes are very similar, divergences are also now increasingly evident. As an example, the recently cloned human mitochondrial DNA polymerase beta-subunit has no counterpart in yeast. Yet, yeast Abf2p and human mtTFA are probably not as closely functionally related as thought previously. Some mtDNA metabolism factors, like DNA ligases, were until recently largely uncharacterized, and have been found to be derived from alternative nuclear products. Many factors involved in the metabolism of mitochondrial DNA are linked through genetic or biochemical interconnections. These links are presented on a map. Finally, we discuss recent studies suggesting that the yeast mtDNA replication system diverges from that observed in man, and may involve recombination, possibly coupled to alternative replication mechanisms like rolling circle replication.

Solution-Phase Processes of Macromolecular Crystallization

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Minamitani, Elizabeth Forsythe

2004-01-01

We have proposed, for the tetragonal form of chicken egg lysozyme, that solution phase assembly processes are needed to form the growth units for crystal nucleation and growth. The starting point for the self-association process is the monomeric protein, and the final crystallographic symmetry is defined by the initial dimerization interactions of the monomers and subsequent n-mers formed, which in turn are a function of the crystallization conditions. It has been suggested that multimeric proteins generally incorporate the underlying multimers symmetry into the final crystallographic symmetry. We posed the question of what happens to a protein that is known to grow as an n-mer when it is placed in solution conditions where it is monomeric. The trypsin-treated, or cut, form of the protein canavalin (CCAN) has been shown to nucleate and grow crystals as a trimer from neutral to slightly acidic solutions. Under these conditions the solution is composed almost wholly of trimers. The insoluble protein can be readily dissolved by weakly basic solution, which results in a solution that is monomeric. There are three possible outcomes to an attempt at crystallization of the protein under monomeric (high pH) conditions: 1) we will obtain the same crystals as under trimer conditions, but at different protein concentrations governed by the self association equilibria; 2) we will obtain crystals having a different symmetry, based upon a monomeric growth unit; 3) we will not obtain crystals. Obtaining the first result would be indicative that the solution-phase self-association process is critical to the crystal nucleation and growth process. The second result would be less clear, as it may also reflect a pH-dependent shift in the trimer-trimer molecular interactions. The third result, particularly for experiments in the transition pH's between trimeric and monomeric CCAN, would indicate that the monomer does not crystallize, and that solution phase self association is not part of the crystal nucleation and growth path. Results are presented for crystallization experiments of CCAN over the pH 6.8 to 9.6 range.

Cooperativity among Rev-Associated Nuclear Export Signals Regulates HIV-1 Gene Expression and Is a Determinant of Virus Species Tropism

PubMed Central

Aligeti, Mounavya; Behrens, Ryan T.; Pocock, Ginger M.; Schindelin, Johannes; Dietz, Christian; Eliceiri, Kevin W.; Swanson, Chad M.; Malim, Michael H.; Ahlquist, Paul

2014-01-01

ABSTRACT Murine cells exhibit a profound block to HIV-1 virion production that was recently mapped to a species-specific structural attribute of the murine version of the chromosomal region maintenance 1 (mCRM1) nuclear export receptor and rescued by the expression of human CRM1 (hCRM1). In human cells, the HIV-1 Rev protein recruits hCRM1 to intron-containing viral mRNAs encoding the Rev response element (RRE), thereby facilitating viral late gene expression. Here we exploited murine 3T3 fibroblasts as a gain-of-function system to study hCRM1's species-specific role in regulating Rev's effector functions. We show that Rev is rapidly exported from the nucleus by mCRM1 despite only weak contributions to HIV-1's posttranscriptional stages. Indeed, Rev preferentially accumulates in the cytoplasm of murine 3T3 cells with or without hCRM1 expression, in contrast to human HeLa cells, where Rev exhibits striking en masse transitions between the nuclear and cytoplasmic compartments. Efforts to bias Rev's trafficking either into or out of the nucleus revealed that Rev encoding a second CRM1 binding domain (Rev-2xNES) or Rev-dependent viral gag-pol mRNAs bearing tandem RREs (GP-2xRRE), rescue virus particle production in murine cells even in the absence of hCRM1. Combined, these results suggest a model wherein Rev-associated nuclear export signals cooperate to regulate the number or quality of CRM1's interactions with viral Rev/RRE ribonucleoprotein complexes in the nucleus. This mechanism regulates CRM1-dependent viral gene expression and is a determinant of HIV-1's capacity to produce virions in nonhuman cell types. IMPORTANCE Cells derived from mice and other nonhuman species exhibit profound blocks to HIV-1 replication. Here we elucidate a block to HIV-1 gene expression attributable to the murine version of the CRM1 (mCRM1) nuclear export receptor. In human cells, hCRM1 regulates the nuclear export of viral intron-containing mRNAs through the activity of the viral Rev adapter protein that forms a multimeric complex on these mRNAs prior to recruiting hCRM1. We demonstrate that Rev-dependent gene expression is poor in murine cells despite the finding that, surprisingly, the bulk of Rev interacts efficiently with mCRM1 and is rapidly exported from the nucleus. Instead, we map the mCRM1 defect to the apparent inability of this factor to engage Rev multimers in the context of large viral Rev/RNA ribonucleoprotein complexes. These findings shed new light on HIV-1 gene regulation and could inform the development of novel antiviral strategies that target viral gene expression. PMID:25275125

Mechanisms Underlying the Formation of Complexes between Maize Starch and Lipids.

PubMed

Chao, Chen; Yu, Jinglin; Wang, Shuo; Copeland, Les; Wang, Shujun

2018-01-10

This study aimed to reveal the mechanism of formation of complexes between native maize starch (NMS) and different types of lipids, namely palmitic acid (PA), monopalmitate glycerol (MPG), dipalmitate glycerol (DPG), and tripalmitate glycerol (TPG). The complexing index followed the order of MPG (96.3%) > PA (41.8%) > TPG (8.3%) > DPG (1.1%), indicating that MPG formed more complexes with NMS than PA, and that few complexes were formed between NMS and DPG and TPG. The NMS-PA complex presented higher thermal transition temperatures and lower enthalpy change than the NMS-MPG complex, indicating that although MPG formed more starch complexes, they had less stable crystalline structures than the complex between NMS and PA. X-ray diffraction (XRD) and Raman spectroscopy showed that both MPG and PA formed V-type crystalline structures with NMS, and confirmed that no complexes were formed between NMS and DPG and TPG. We conclude that the monoglyceride formed more starch-lipid complex with maize starch than PA, but that the monoglyceride complex had a less stable structure than that formed with PA. The di- and triglycerides did not form complexes with maize starch.

Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It

PubMed Central

Virreira Winter, Sebastian; Niedelman, Wendy; Jensen, Kirk D.; Rosowski, Emily E.; Julien, Lindsay; Spooner, Eric; Caradonna, Kacey; Burleigh, Barbara A.; Saeij, Jeroen P. J.; Ploegh, Hidde L.; Frickel, Eva-Maria

2011-01-01

IFN-γ is a major cytokine that mediates resistance against the intracellular parasite Toxoplasma gondii. The p65 guanylate-binding proteins (GBPs) are strongly induced by IFN-γ. We studied the behavior of murine GBP1 (mGBP1) upon infection with T. gondii in vitro and confirmed that IFN-γ-dependent re-localization of mGBP1 to the parasitophorous vacuole (PV) correlates with the virulence type of the parasite. We identified three parasitic factors, ROP16, ROP18, and GRA15 that determine strain-specific accumulation of mGBP1 on the PV. These highly polymorphic proteins are held responsible for a large part of the strain-specific differences in virulence. Therefore, our data suggest that virulence of T. gondii in animals may rely in part on recognition by GBPs. However, phagosomes or vacuoles containing Trypanosoma cruzi did not recruit mGBP1. Co-immunoprecipitation revealed mGBP2, mGBP4, and mGBP5 as binding partners of mGBP1. Indeed, mGBP2 and mGBP5 co-localize with mGBP1 in T. gondii-infected cells. T. gondii thus elicits a cell-autonomous immune response in mice with GBPs involved. Three parasitic virulence factors and unknown IFN-γ-dependent host factors regulate this complex process. Depending on the virulence of the strains involved, numerous GBPs are brought to the PV as part of a large, multimeric structure to combat T. gondii. PMID:21931713

Cooling-induced SUMOylation of EXOSC10 down-regulates ribosome biogenesis

PubMed Central

Bastide, Amandine; Peretti, Diego; Roobol, Anne; Roobol, Jo; Mallucci, Giovanna R.; Smales, C. Mark; Willis, Anne E.

2016-01-01

The RNA exosome is essential for 3′ processing of functional RNA species and degradation of aberrant RNAs in eukaryotic cells. Recent reports have defined the substrates of the exosome catalytic domains and solved the multimeric structure of the exosome complex. However, regulation of exosome activity remains poorly characterized, especially in response to physiological stress. Following the observation that cooling of mammalian cells results in a reduction in 40S:60S ribosomal subunit ratio, we uncover regulation of the nuclear exosome as a result of reduced temperature. Using human cells and an in vivo model system allowing whole-body cooling, we observe reduced EXOSC10 (hRrp6, Pm/Scl-100) expression in the cold. In parallel, both models of cooling increase global SUMOylation, leading to the identification of specific conjugation of SUMO1 to EXOSC10, a process that is increased by cooling. Furthermore, we define the major SUMOylation sites in EXOSC10 by mutagenesis and show that overexpression of SUMO1 alone is sufficient to suppress EXOSC10 abundance. Reducing EXOSC10 expression by RNAi in human cells correlates with the 3′ preribosomal RNA processing defects seen in the cold as well as reducing the 40S:60S ratio, a previously uncharacterized consequence of EXOSC10 suppression. Together, this work illustrates that EXOSC10 can be modified by SUMOylation and identifies a physiological stress where this regulation is prevalent both in vitro and in vivo. PMID:26857222

PrP(C) regulates epidermal growth factor receptor function and cell shape dynamics in Neuro2a cells.

PubMed

Llorens, Franc; Carulla, Patricia; Villa, Ana; Torres, Juan M; Fortes, Puri; Ferrer, Isidre; del Río, José A

2013-10-01

The prion protein (PrP) plays a key role in prion disease pathogenesis. Although the misfolded and pathologic variant of this protein (PrP(SC)) has been studied in depth, the physiological role of PrP(C) remains elusive and controversial. PrP(C) is a cell-surface glycoprotein involved in multiple cellular functions at the plasma membrane, where it interacts with a myriad of partners and regulates several intracellular signal transduction cascades. However, little is known about the gene expression changes modulated by PrP(C) in animals and in cellular models. In this article, we present PrP(C)-dependent gene expression signature in N2a cells and its implication in the most overrepresented functions: cell cycle, cell growth and proliferation, and maintenance of cell shape. PrP(C) over-expression enhances cell proliferation and cell cycle re-entrance after serum stimulation, while PrP(C) silencing slows down cell cycle progression. In addition, MAP kinase and protein kinase B (AKT) pathway activation are under the regulation of PrP(C) in asynchronous cells and following mitogenic stimulation. These effects are due in part to the modulation of epidermal growth factor receptor (EGFR) by PrP(C) in the plasma membrane, where the two proteins interact in a multimeric complex. We also describe how PrP(C) over-expression modulates filopodia formation by Rho GTPase regulation mainly in an AKT-Cdc42-N-WASP-dependent pathway. © 2013 International Society for Neurochemistry.

Identification of critical regions in human SAMHD1 required for nuclear localization and Vpx-mediated degradation.

PubMed

Guo, Haoran; Wei, Wei; Wei, Zhenhong; Liu, Xianjun; Evans, Sean L; Yang, Weiming; Wang, Hong; Guo, Ying; Zhao, Ke; Zhou, Jian-Ying; Yu, Xiao-Fang

2013-01-01

The sterile alpha motif (SAM) and HD domain-containing protein-1 (SAMHD1) inhibits the infection of resting CD4+ T cells and myeloid cells by human and related simian immunodeficiency viruses (HIV and SIV). Vpx inactivates SAMHD1 by promoting its proteasome-dependent degradation through an interaction with CRL4 (DCAF1) E3 ubiquitin ligase and the C-terminal region of SAMHD1. However, the determinants in SAMHD1 that are required for Vpx-mediated degradation have not been well characterized. SAMHD1 contains a classical nuclear localization signal (NLS), and NLS point mutants are cytoplasmic and resistant to Vpx-mediated degradation. Here, we demonstrate that NLS-mutant SAMHD1 K11A can be rescued by wild-type SAMHD1, restoring its nuclear localization; consequently, SAMHD1 K11A became sensitive to Vpx-mediated degradation in the presence of wild-type SAMHD1. Surprisingly, deletion of N-terminal regions of SAMHD1, including the classical NLS, generated mutant SAMHD1 proteins that were again sensitive to Vpx-mediated degradation. Unlike SAMHD1 K11A, these deletion mutants could be detected in the nucleus. Interestingly, NLS-defective SAMHD1 could still bind to karyopherin-β1 and other nuclear proteins. We also determined that the linker region between the SAM and HD domain and the HD domain itself is important for Vpx-mediated degradation but not Vpx interaction. Thus, SAMHD1 contains an additional nuclear targeting mechanism in addition to the classical NLS. Our data indicate that multiple regions in SAMHD1 are critical for Vpx-mediated nuclear degradation and that association with Vpx is not sufficient for Vpx-mediated degradation of SAMHD1. Since the linker region and HD domain may be involved in SAMHD1 multimerization, our results suggest that SAMHD1 multimerization may be required for Vpx-mediation degradation.

The effect of multimeric adiponectin isoforms and leptin on the function of rheumatoid fibroblast-like synoviocytes.

PubMed

Kontny, E; Janicka, I; Skalska, U; Maśliński, W

2015-01-01

To evaluate the effects of physiologically relevant concentrations of multimeric adiponectin isoforms and leptin on the function of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). FLS, isolated from the synovial tissue of 21 RA patients, were stimulated for 24 h with interleukin (IL)-1β (1 ng/mL) and adiponectin isoforms [fraction enriched with high-molecular-weight (HMW) oligomers and middle-molecular-weight (MMW) hexamers or low-molecular-weight (LMW) trimers, 10 μg/mL each], or leptin (10 ng/mL), either separately or in a combination of IL-1β and the respective adipokine. Moreover, cells were pre-treated for 24 h with adipokines, then stimulated for 8 h with IL-1β. The concentrations of IL-6, IL-8, matrix metalloproteinase (MMP)-3, and dickkopf (DKK)-1, an inhibitor of osteoblastogenesis, in culture supernatants, as well as the concentrations of leptin, HMW, MMW, and LMW adiponectin in sera and synovial fluid (SF) samples, were measured by specific enzyme-linked immunosorbent assays (ELISAs). In comparison with sera, SF samples contained similar amounts of leptin, lower amounts of total adiponectin but a higher proportion of the LMW isoform. Separately added IL-1β and HMW/MMW adiponectin, but not LMW adiponectin or leptin, up-regulated the release of IL-6, IL-8, and MMP-3 from FLS but no synergy was observed in co-stimulation experiments. However, pre-treatment of FLS with HMW/MMW or LMW significantly raised the IL-1β-triggered secretion of MMP-3 and IL-6 or MMP-3, respectively. Adiponectin not only triggers pro-inflammatory and pro-destructive activities of rheumatoid FLS but also pre-disposes these cells to a stronger response to IL-1β. Thus, it is likely that adiponectin is more important in the initiation phase than in the chronic phase of RA.

The sigma-1 receptor modulates dopamine transporter conformation and cocaine binding and may thereby potentiate cocaine self-administration in rats.

PubMed

Hong, Weimin Conrad; Yano, Hideaki; Hiranita, Takato; Chin, Frederick T; McCurdy, Christopher R; Su, Tsung-Ping; Amara, Susan G; Katz, Jonathan L

2017-07-07

The dopamine transporter (DAT) regulates dopamine (DA) neurotransmission by recapturing DA into the presynaptic terminals and is a principal target of the psychostimulant cocaine. The sigma-1 receptor (σ 1 R) is a molecular chaperone, and its ligands have been shown to modulate DA neuronal signaling, although their effects on DAT activity are unclear. Here, we report that the prototypical σ 1 R agonist (+)-pentazocine potentiated the dose response of cocaine self-administration in rats, consistent with the effects of the σR agonists PRE-084 and DTG (1,3-di- o -tolylguanidine) reported previously. These behavioral effects appeared to be correlated with functional changes of DAT. Preincubation with (+)-pentazocine or PRE-084 increased the B max values of [ 3 H]WIN35428 binding to DAT in rat striatal synaptosomes and transfected cells. A specific interaction between σ 1 R and DAT was detected by co-immunoprecipitation and bioluminescence resonance energy transfer assays. Mutational analyses indicated that the transmembrane domain of σ 1 R likely mediated this interaction. Furthermore, cysteine accessibility assays showed that σ 1 R agonist preincubation potentiated cocaine-induced changes in DAT conformation, which were blocked by the specific σ 1 R antagonist CM304. Moreover, σ 1 R ligands had distinct effects on σ 1 R multimerization. CM304 increased the proportion of multimeric σ 1 Rs, whereas (+)-pentazocine increased monomeric σ 1 Rs. Together these results support the hypothesis that σ 1 R agonists promote dissociation of σ 1 R multimers into monomers, which then interact with DAT to stabilize an outward-facing DAT conformation and enhance cocaine binding. We propose that this novel molecular mechanism underlies the behavioral potentiation of cocaine self-administration by σ 1 R agonists in animal models. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The proto-oncogene product p120CBL and the adaptor proteins CRKL and c-CRK link c-ABL, p190BCR/ABL and p210BCR/ABL to the phosphatidylinositol-3' kinase pathway.

PubMed

Sattler, M; Salgia, R; Okuda, K; Uemura, N; Durstin, M A; Pisick, E; Xu, G; Li, J L; Prasad, K V; Griffin, J D

1996-02-15

Chronic myelogenous leukemia (CML) and some acute lymphoblastic leukemias (ALL) are caused by the t(9;22) chromosome translocation, which produces the constitutively activated BCR/ABL tyrosine kinase. When introduced into factor dependent hematopoietic cell lines, BCR/ABL induces the tyrosine phosphorylation of many cellular proteins. One prominent BCR/ABL substrate is p120CBL, the cellular homolog of the v-Cbl oncoprotein. In an effort to understand the possible contribution of p120CBL to transformation by BCR/ABL, we looked for cellular proteins which associate with p120CBL in hematopoietic cell lines transformed by BCR/ABL. In addition to p210BCR/ABL and c-ABL, p120CBL coprecipitated with an 85 kDa phosphoprotein, which was identified as the p85 subunit of PI3K. Anti-p120CBL immunoprecipitates from BCR/ABL-transformed, but not from untransformed, cell lines contained PI3K lipid kinase activity. Interestingly, the adaptor proteins CRKL and c-CRK were also found in these complexes. In vitro binding studies indicated that the SH2 domains of CRKL and c-CRK bound directly to p120CBL, while the SH3 domains of c-CRK and CRKL bound to BCR/ABL and c-ABL. The N-terminal and the C-terminal SH2 and the SH3 domain of p85PI3K bound directly in vitro to p120CBL. The ABL-SH2, but not ABL-SH3, could also bind to p120CBL. These data suggest that BCR/ABL may induce the formation of multimeric complexes of signaling proteins which include p120CBL, PI3K, c-CRK or CRKL, c-ABL and BCR/ABL itself.

Convergent evolution of chromatin modification by structurally distinct enzymes: comparative enzymology of histone H3 Lys²⁷ methylation by human polycomb repressive complex 2 and vSET.

PubMed

Swalm, Brooke M; Hallenbeck, Kenneth K; Majer, Christina R; Jin, Lei; Scott, Margaret Porter; Moyer, Mikel P; Copeland, Robert A; Wigle, Tim J

2013-07-15

H3K27 (histone H3 Lys27) methylation is an important epigenetic modification that regulates gene transcription. In humans, EZH (enhancer of zeste homologue) 1 and EZH2 are the only enzymes capable of catalysing methylation of H3K27. There is great interest in understanding structure-function relationships for EZH2, as genetic alterations in this enzyme are thought to play a causal role in a number of human cancers. EZH2 is challenging to study because it is only active in the context of the multi-subunit PRC2 (polycomb repressive complex 2). vSET is a viral lysine methyltransferase that represents the smallest protein unit capable of catalysing H3K27 methylation. The crystal structure of this minimal catalytic protein has been solved and researchers have suggested that vSET might prove useful as an EZH2 surrogate for the development of active site-directed inhibitors. To test this proposition, we conducted comparative enzymatic analysis of human EZH2 and vSET and report that, although both enzymes share similar preferences for methylation of H3K27, they diverge in terms of their permissiveness for catalysing methylation of alternative histone lysine sites, their relative preferences for utilization of multimeric macromolecular substrates, their active site primary sequences and, most importantly, their sensitivity to inhibition by drug-like small molecules. The cumulative data led us to suggest that EZH2 and vSET have very distinct active site structures, despite the commonality of the reaction catalysed by the two enzymes. Hence, the EZH2 and vSET pair of enzymes represent an example of convergent evolution in which distinct structural solutions have developed to solve a common catalytic need.

Linker-mediated assembly of gold nanoparticles into multimeric motifs

NASA Astrophysics Data System (ADS)

Sikora, Mateusz; Szymczak, Piotr; Thompson, Damien; Cieplak, Marek

2011-11-01

We present a theoretical description of linker-mediated self-assembly of gold nanoparticles (Au-NP). Using mesoscale simulations with a coarse-grained model for the Au NPs and dirhenium-based linker molecules, we investigate the conditions under which large clusters can grow and construct a phase diagram that identifies favorable growth conditions in terms of floating and bound linker concentrations. The findings can be considered as generic, as we expect other NP-linker systems to behave in a qualitatively similar way. In particular, we also discuss the case of antibody-functionalised Au NPs connected by the C-reactive proteins (CRPs). We extract some general rules for NP linking that may aid the production of size- and shape-specific NP clusters for technology applications.

Implication of a Central Cysteine Residue and the HHCC Domain of Moloney Murine Leukemia Virus Integrase Protein in Functional Multimerization

PubMed Central

Donzella, George A.; Leon, Oscar; Roth, Monica J.

1998-01-01

Moloney murine leukemia virus (M-MuLV) IN-IN protein interactions important for catalysis of strand transfer and unimolecular and bimolecular disintegration reactions were investigated by using a panel of chemically modified M-MuLV IN proteins. Functional complementation of an HHCC-deleted protein (NΔ105) by an independent HHCC domain (CΔ232) was severely compromised by NEM modification of either subunit. Productive NΔ105 IN-DNA interactions with a disintegration substrate lacking a long terminal repeat 5′-single-stranded tail also required complementation by a functional HHCC domain. Virus encoding the C209A M-MuLV IN mutation exhibited delayed virion production and replication kinetics. PMID:9445080

CABS-flex 2.0: a web server for fast simulations of flexibility of protein structures.

PubMed

Kuriata, Aleksander; Gierut, Aleksandra Maria; Oleniecki, Tymoteusz; Ciemny, Maciej Pawel; Kolinski, Andrzej; Kurcinski, Mateusz; Kmiecik, Sebastian

2018-05-14

Classical simulations of protein flexibility remain computationally expensive, especially for large proteins. A few years ago, we developed a fast method for predicting protein structure fluctuations that uses a single protein model as the input. The method has been made available as the CABS-flex web server and applied in numerous studies of protein structure-function relationships. Here, we present a major update of the CABS-flex web server to version 2.0. The new features include: extension of the method to significantly larger and multimeric proteins, customizable distance restraints and simulation parameters, contact maps and a new, enhanced web server interface. CABS-flex 2.0 is freely available at http://biocomp.chem.uw.edu.pl/CABSflex2.

Hemocyanin gene family evolution in spiders (Araneae), with implications for phylogenetic relationships and divergence times in the infraorder Mygalomorphae.

PubMed

Starrett, James; Hedin, Marshal; Ayoub, Nadia; Hayashi, Cheryl Y

2013-07-25

Hemocyanins are multimeric copper-containing hemolymph proteins involved in oxygen binding and transport in all major arthropod lineages. Most arachnids have seven primary subunits (encoded by paralogous genes a-g), which combine to form a 24-mer (4×6) quaternary structure. Within some spider lineages, however, hemocyanin evolution has been a dynamic process with extensive paralog duplication and loss. We have obtained hemocyanin gene sequences from numerous representatives of the spider infraorders Mygalomorphae and Araneomorphae in order to infer the evolution of the hemocyanin gene family and estimate spider relationships using these conserved loci. Our hemocyanin gene tree is largely consistent with the previous hypotheses of paralog relationships based on immunological studies, but reveals some discrepancies in which paralog types have been lost or duplicated in specific spider lineages. Analyses of concatenated hemocyanin sequences resolved deep nodes in the spider phylogeny and recovered a number of clades that are supported by other molecular studies, particularly for mygalomorph taxa. The concatenated data set is also used to estimate dates of higher-level spider divergences and suggests that the diversification of extant mygalomorphs preceded that of extant araneomorphs. Spiders are diverse in behavior and respiratory morphology, and our results are beneficial for comparative analyses of spider respiration. Lastly, the conserved hemocyanin sequences allow for the inference of spider relationships and ancient divergence dates. Copyright © 2013 Elsevier B.V. All rights reserved.

Satellite RNAs of plant viruses: structures and biological effects.

PubMed Central

Roossinck, M J; Sleat, D; Palukaitis, P

1992-01-01

Plant viruses often contain parasites of their own, referred to as satellites. Satellite RNAs are dependent on their associated (helper) virus for both replication and encapsidation. Satellite RNAs vary from 194 to approximately 1,500 nucleotides (nt). The larger satellites (900 to 1,500 nt) contain open reading frames and express proteins in vitro and in vivo, whereas the smaller satellites (194 to 700 nt) do not appear to produce functional proteins. The smaller satellites contain a high degree of secondary structure involving 49 to 73% of their sequences, with the circular satellites containing more base pairing than the linear satellites. Many of the smaller satellites produce multimeric forms during replication. There are various models to account for their formation and role in satellite replication. Some of these smaller satellites encode ribozymes and are able to undergo autocatalytic cleavage. The enzymology of satellite replication is poorly understood, as is the replication of their helper viruses. In many cases the coreplication of satellites suppresses the replication of the helper virus genome. This is usually paralleled by a reduction in the disease induced by the helper virus; however, there are notable exceptions in which the satellite exacerbates the pathogenicity of the helper virus, albeit on only a limited number of hosts. The ameliorative satellites are being assessed as biocontrol agents of virus-induced disease. In greenhouse studies, satellites have been known to "spontaneously" appear in virus cultures. The possible origin of satellites will be briefly considered. PMID:1620065

Behavior of lysozyme adsorbed onto biological liquid crystal lipid monolayer at the air/water interface

NASA Astrophysics Data System (ADS)

Lu, Xiaolong; Shi, Ruixin; Hao, Changchun; Chen, Huan; Zhang, Lei; Li, Junhua; Xu, Guoqing; Sun, Runguang

2016-09-01

The interaction between proteins and lipids is one of the basic problems of modern biochemistry and biophysics. The purpose of this study is to compare the penetration degree of lysozyme into 1,2-diapalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethano-lamine (DPPE) by analyzing the data of surface pressure-area (π-A) isotherms and surface pressure-time (π-T) curves. Lysozyme can penetrate into both DPPC and DPPE monolayers because of the increase of surface pressure at an initial pressure of 15 mN/m. However, the changes of DPPE are larger than DPPC, indicating stronger interaction of lysozyme with DPPE than DPPC. The reason may be due to the different head groups and phase state of DPPC and DPPE monolayers at the surface pressure of 15 mN/m. Atomic force microscopy reveals that lysozyme was absorbed by DPPC and DPPE monolayers, which leads to self-aggregation and self-assembly, forming irregular multimers and conical multimeric. Through analysis, we think that the process of polymer formation is similar to the aggregation mechanism of amyloid fibers. Project supported by the National Natural Science Foundation of China (Grant Nos. 21402114 and 11544009), the Natural Science Basic Research Plan in Shaanxi Province of China (Grant No. 2016JM2010), the Fundamental Research Funds for the Central Universities of China (Grant No. GK201603026), and the National University Science and Technology Innovation Project of China (Grant No. 201610718013).

Functional Importance of Covalent Homodimer of Reelin Protein Linked via Its Central Region*

PubMed Central

Yasui, Norihisa; Kitago, Yu; Beppu, Ayako; Kohno, Takao; Morishita, Shunsuke; Gomi, Hiroki; Nagae, Masamichi; Hattori, Mitsuharu; Takagi, Junichi

2011-01-01

Reelin is a 3461-residue secreted glycoprotein that plays a critical role in brain development through its action on target neurons. Although it is known that functional reelin protein exists as multimer formed by interchain disulfide bond(s) as well as through non-covalent interactions, the chemical nature of the multimer assembly has been elusive. In the present study, we identified, among 122 cysteines present in full-length reelin, the single critical cysteine residue (Cys2101) responsible for the covalent multimerization. C2101A mutant reelin failed to assemble into disulfide-bonded multimers, whereas it still exhibited non-covalently associated high molecular weight oligomeric states in solution. Detailed analysis of tryptic fragments produced from the purified reelin proteins revealed that the minimum unit of the multimer is a homodimeric reelin linked via Cys2101 present in the central region and that this cysteine does not connect to the N-terminal region of reelin, which had been postulated as the primary oligomerization domain. A surface plasmon resonance binding assay confirmed that C2101A mutant reelin retained binding capability toward two neuronal receptors apolipoprotein E receptor 2 and very low density lipoprotein receptor. However, it failed to show signaling activity in the assay using the cultured neurons. These results indicate that an intact higher order architecture of reelin multimer maintained by both Cys2101-mediated homodimerization and other non-covalent association present elsewhere in the reelin primary structure are essential for exerting its full biological activity. PMID:21844191

Mycobacterium tuberculosis Hip1 modulates macrophage responses through proteolysis of GroEL2.

PubMed

Naffin-Olivos, Jacqueline L; Georgieva, Maria; Goldfarb, Nathan; Madan-Lala, Ranjna; Dong, Lauren; Bizzell, Erica; Valinetz, Ethan; Brandt, Gabriel S; Yu, Sarah; Shabashvili, Daniil E; Ringe, Dagmar; Dunn, Ben M; Petsko, Gregory A; Rengarajan, Jyothi

2014-05-01

Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.

Mycobacterium tuberculosis Hip1 Modulates Macrophage Responses through Proteolysis of GroEL2

PubMed Central

Madan-Lala, Ranjna; Dong, Lauren; Bizzell, Erica; Valinetz, Ethan; Brandt, Gabriel S.; Yu, Sarah; Shabashvili, Daniil E.; Ringe, Dagmar; Dunn, Ben M.; Petsko, Gregory A.; Rengarajan, Jyothi

2014-01-01

Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb. PMID:24830429

TLR-adjuvanted nanoparticle vaccines differentially influence the quality and longevity of responses to malaria antigen Pfs25.

PubMed

Thompson, Elizabeth A; Ols, Sebastian; Miura, Kazutoyo; Rausch, Kelly; Narum, David L; Spångberg, Mats; Juraska, Michal; Wille-Reece, Ulrike; Weiner, Amy; Howard, Randall F; Long, Carole A; Duffy, Patrick E; Johnston, Lloyd; O'Neil, Conlin P; Loré, Karin

2018-05-17

Transmission-blocking vaccines (TBVs) are considered an integral element of malaria eradication efforts. Despite promising evaluations of Plasmodium falciparum Pfs25-based TBVs in mice, clinical trials have failed to induce robust and long-lived Ab titers, in part due to the poorly immunogenic nature of Pfs25. Using nonhuman primates, we demonstrate that multiple aspects of Pfs25 immunity were enhanced by antigen encapsulation in poly(lactic-co-glycolic acid)-based [(PLGA)-based] synthetic vaccine particles (SVP[Pfs25]) and potent TLR-based adjuvants. SVP[Pfs25] increased Ab titers, Pfs25-specific plasmablasts, circulating memory B cells, and plasma cells in the bone marrow when benchmarked against the clinically tested multimeric form Pfs25-EPA given with GLA-LSQ. SVP[Pfs25] also induced the first reported Pfs25-specific circulating Th1 and Tfh cells to our knowledge. Multivariate correlative analysis indicated several mechanisms for the improved Ab responses. While Pfs25-specific B cells were responsible for increasing Ab titers, T cell responses stimulated increased Ab avidity. The innate immune activation differentially stimulated by the adjuvants revealed a strong correlation between type I IFN polarization, induced by R848 and CpG, and increased Ab half-life and longevity. Collectively, the data identify ways to improve vaccine-induced immunity to poorly immunogenic proteins, both by the choice of antigen and adjuvant formulation, and highlight underlying immunological mechanisms.

Replication of avocado sunblotch viroid: evidence for a symmetric pathway with two rolling circles and hammerhead ribozyme processing.

PubMed Central

Daròs, J A; Marcos, J F; Hernández, C; Flores, R

1994-01-01

The structure of a series of RNAs extracted from avocado infected by the 247-nt avocado sunblotch viroid (ASBVd) was investigated. The identification of multistranded complexes containing circular ASBVd RNAs of (+) and (-) polarity suggests that replication of ASBVd proceeds through a symmetric pathway with two rolling circles where these two circular RNAs are the templates. This is in contrast to the replication of potato spindle tuber viroid and probably of most of its related viroids, which proceeds via an asymmetric pathway where circular (+)-strand and linear multimeric (-)-strand RNAs are the two templates. Linear (+) and (-) ASBVd RNAs of subgenomic length (137 nt and about 148 nt, respectively) and one linear (+)-strand ASBVd RNA of supragenomic length (383-384 nt) were also found in viroid-infected tissue. The two linear (+)-strand RNAs have the same 5'- and 3'-terminal sequences, with the supragenomic species being a fusion product of the monomeric and subgenomic (+)-strand ASBVd RNAs. The 3' termini of these two (+)-strand molecules, which at least in the subgenomic RNA has an extra nontemplate cytidylate residue, could represent sites of either premature termination of the (+)-strands or specific initiation of the (-)-strands. The 5' termini of sub- and supragenomic (+)-strand and the 5' terminus of the subgenomic (-)-strand ASBVd RNA are identical to those produced in the in vitro self-cleavage reactions of (+) and (-) dimeric ASBVd RNAs, respectively. These observations strongly suggest that the hammerhead structures which mediate the in vitro self-cleavage reactions are also operative in vivo. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:7809126

Mcm3 replicative helicase mutation impairs neuroblast proliferation and memory in Drosophila.

PubMed

Blumröder, R; Glunz, A; Dunkelberger, B S; Serway, C N; Berger, C; Mentzel, B; de Belle, J S; Raabe, T

2016-09-01

In the developing Drosophila brain, a small number of neural progenitor cells (neuroblasts) generate in a co-ordinated manner a high variety of neuronal cells by integration of temporal, spatial and cell-intrinsic information. In this study, we performed the molecular and phenotypic characterization of a structural brain mutant called small mushroom bodies (smu), which was isolated in a screen for mutants with altered brain structure. Focusing on the mushroom body neuroblast lineages we show that failure of neuroblasts to generate the normal number of mushroom body neurons (Kenyon cells) is the major cause of the smu phenotype. In particular, the premature loss of mushroom body neuroblasts caused a pronounced effect on the number of late-born Kenyon cells. Neuroblasts showed no obvious defects in processes controlling asymmetric cell division, but generated less ganglion mother cells. Cloning of smu uncovered a single amino acid substitution in an evolutionarily conserved protein interaction domain of the Minichromosome maintenance 3 (Mcm3) protein. Mcm3 is part of the multimeric Cdc45/Mcm/GINS (CMG) complex, which functions as a helicase during DNA replication. We propose that at least in the case of mushroom body neuroblasts, timely replication is not only required for continuous proliferation but also for their survival. The absence of Kenyon cells in smu reduced learning and early phases of conditioned olfactory memory. Corresponding to the absence of late-born Kenyon cells projecting to α'/β' and α/β lobes, smu is profoundly defective in later phases of persistent memory. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment.

PubMed

Iezzi, María Elena; Policastro, Lucía; Werbajh, Santiago; Podhajcer, Osvaldo; Canziani, Gabriela Alicia

2018-01-01

Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs), in particular those engineered from the variable heavy-chain fragment (VHH gene) found in Camelidae heavy-chain antibodies (or IgG2 and IgG3), are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR) fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition "modules" to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo . Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads.

Molecular Modeling and Imaging of Initial Stages of Cellulose Fibril Assembly: Evidence for a Disordered Intermediate Stage

PubMed Central

Haigler, Candace H.; Grimson, Mark J.; Gervais, Julien; Le Moigne, Nicolas; Höfte, Herman; Monasse, Bernard; Navard, Patrick

2014-01-01

The remarkable mechanical strength of cellulose reflects the arrangement of multiple β-1,4-linked glucan chains in a para-crystalline fibril. During plant cellulose biosynthesis, a multimeric cellulose synthesis complex (CSC) moves within the plane of the plasma membrane as many glucan chains are synthesized from the same end and in close proximity. Many questions remain about the mechanism of cellulose fibril assembly, for example must multiple catalytic subunits within one CSC polymerize cellulose at the same rate? How does the cellulose fibril bend to align horizontally with the cell wall? Here we used mathematical modeling to investigate the interactions between glucan chains immediately after extrusion on the plasma membrane surface. Molecular dynamics simulations on groups of six glucans, each originating from a position approximating its extrusion site, revealed initial formation of an uncrystallized aggregate of chains from which a protofibril arose spontaneously through a ratchet mechanism involving hydrogen bonds and van der Waals interactions between glucose monomers. Consistent with the predictions from the model, freeze-fracture transmission electron microscopy using improved methods revealed a hemispherical accumulation of material at points of origination of apparent cellulose fibrils on the external surface of the plasma membrane where rosette-type CSCs were also observed. Together the data support the possibility that a zone of uncrystallized chains on the plasma membrane surface buffers the predicted variable rates of cellulose polymerization from multiple catalytic subunits within the CSC and acts as a flexible hinge allowing the horizontal alignment of the crystalline cellulose fibrils relative to the cell wall. PMID:24722535

Oligomerization of GPCRs involved in endocrine regulation.

PubMed

Kleinau, Gunnar; Müller, Anne; Biebermann, Heike

2016-07-01

More than 800 different human membrane-spanning G-protein-coupled receptors (GPCRs) serve as signal transducers at biological barriers. These receptors are activated by a wide variety of ligands such as peptides, ions and hormones, and are able to activate a diverse set of intracellular signaling pathways. GPCRs are of central importance in endocrine regulation, which underpins the significance of comprehensively studying these receptors and interrelated systems. During the last decade, the capacity for multimerization of GPCRs was found to be a common and functionally relevant property. The interaction between GPCR monomers results in higher order complexes such as homomers (identical receptor subtype) or heteromers (different receptor subtypes), which may be present in a specific and dynamic monomer/oligomer equilibrium. It is widely accepted that the oligomerization of GPCRs is a mechanism for determining the fine-tuning and expansion of cellular processes by modification of ligand action, expression levels, and related signaling outcome. Accordingly, oligomerization provides exciting opportunities to optimize pharmacological treatment with respect to receptor target and tissue selectivity or for the development of diagnostic tools. On the other hand, GPCR heteromerization may be a potential reason for the undesired side effects of pharmacological interventions, faced with numerous and common mutual signaling modifications in heteromeric constellations. Finally, detailed deciphering of the physiological occurrence and relevance of specific GPCR/GPCR-ligand interactions poses a future challenge. This review will tackle the aspects of GPCR oligomerization with specific emphasis on family A GPCRs involved in endocrine regulation, whereby only a subset of these receptors will be discussed in detail. © 2016 Society for Endocrinology.

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

Szymańska, Paulina; Martin, Katie R.; MacKeigan, Jeffrey P.

We constructed a mechanistic, computational model for regulation of (macro)autophagy and protein synthesis (at the level of translation). The model was formulated to study the system-level consequences of interactions among the following proteins: two key components of MTOR complex 1 (MTORC1), namely the protein kinase MTOR (mechanistic target of rapamycin) and the scaffold protein RPTOR; the autophagy-initiating protein kinase ULK1; and the multimeric energy-sensing AMP-activated protein kinase (AMPK). Inputs of the model include intrinsic AMPK kinase activity, which is taken as an adjustable surrogate parameter for cellular energy level or AMP:ATP ratio, and rapamycin dose, which controls MTORC1 activity. Outputsmore » of the model include the phosphorylation level of the translational repressor EIF4EBP1, a substrate of MTORC1, and the phosphorylation level of AMBRA1 (activating molecule in BECN1-regulated autophagy), a substrate of ULK1 critical for autophagosome formation. The model incorporates reciprocal regulation of mTORC1 and ULK1 by AMPK, mutual inhibition of MTORC1 and ULK1, and ULK1-mediated negative feedback regulation of AMPK. Through analysis of the model, we find that these processes may be responsible, depending on conditions, for graded responses to stress inputs, for bistable switching between autophagy and protein synthesis, or relaxation oscillations, comprising alternating periods of autophagy and protein synthesis. A sensitivity analysis indicates that the prediction of oscillatory behavior is robust to changes of the parameter values of the model. The model provides testable predictions about the behavior of the AMPK-MTORC1-ULK1 network, which plays a central role in maintaining cellular energy and nutrient homeostasis.« less

Plasma adiponectin complexes have distinct biochemical characteristics.

PubMed

Schraw, Todd; Wang, Zhao V; Halberg, Nils; Hawkins, Meredith; Scherer, Philipp E

2008-05-01

Adipocytes release the secretory protein adiponectin in a number of different higher-order complexes. Once synthesized and assembled in the secretory pathway of the adipocyte, these complexes circulate as biochemically distinct and stable entities with little evidence of interchange between the different forms that include a high-molecular-weight (HMW) species, a hexamer (low-molecular-weight form), and a trimeric form of the complexes. Here, we validate a high-resolution gel filtration method that reproducibly separates the three complexes in recombinant adiponectin and adiponectin from human and murine samples. We demonstrate that the HMW form is prominently reduced in male vs. female subjects and in obese, insulin-resistant vs. lean, insulin-sensitive individuals. A direct comparison of human and mouse adiponectin demonstrates that the trimer is generally more abundant in human serum. Furthermore, when the production of adiponectin is reduced, either by obesity or in mice carrying only a single functional allele of the adiponectin locus, then the amount of the HMW form is selectively reduced in circulation. The complex distribution of adiponectin can be regulated in several ways. Both mouse and human HMW adiponectin are very stable under basic conditions but are exquisitely labile under acidic conditions below pH 7. Murine and human adiponectin HMW forms also display differential susceptibility to the presence of calcium in the buffer. A mutant form of adiponectin unable to bind calcium is less susceptible to changes in calcium concentrations. However, the lack of calcium binding results in a destabilization of the structure. Disulfide bond formation (at position C39) is also important for complex formation. A mutant form of adiponectin lacking C39 prominently forms HMW and trimer but not the low-molecular-weight form. Injection of adiponectin with a fluorescent label reveals that over time, the various complexes do not interconvert in vivo. The stability of adiponectin complexes highlights that the production and secretion of these forms from fat cells has a major influence on the circulating levels of each complex.

Molecular dynamics studies of the protein-protein interactions in inhibitor of κB kinase-β.

PubMed

Jones, Michael R; Liu, Cong; Wilson, Angela K

2014-02-24

Activation of the inhibitor of κB kinase subunit β (IKKβ) oligomer initiates a cascade that results in the translocation of transcription factors involved in mediating immune responses. Dimerization of IKKβ is required for its activation. Coarse-grained and atomistic molecular dynamics simulations were used to investigate the conformation-activity and structure-activity relationships within the oligomer assembly of IKKβ that are impacted upon activation, mutation, and binding of ATP. Intermolecular interactions, free energies, and conformational changes were compared among several conformations, including a monomer, two different dimers, and the tetramer. Modifications to the activation segment induce conformational changes that disrupt dimerization and suggest that the multimeric assembly mediates a global stability for the enzyme that influences the activity of IKKβ.

HIV-1 matrix domain removal ameliorates virus assembly and processing defects incurred by positive nucleocapsid charge elimination.

PubMed

Ko, Li-Jung; Yu, Fu-Hsien; Huang, Kuo-Jung; Wang, Chin-Tien

2015-01-01

Human immunodeficiency virus type 1 nucleocapsid (NC) basic residues presumably contribute to virus assembly via RNA, which serves as a scaffold for Gag-Gag interaction during particle assembly. To determine whether NC basic residues play a role in Gag cleavage (thereby impacting virus assembly), Gag processing efficiency and virus particle production were analyzed for an HIV-1 mutant NC15A, with alanine serving as a substitute for all NC basic residues. Results indicate that NC15A significantly impaired virus maturation in addition to significantly affecting Gag membrane binding and assembly. Interestingly, removal of the matrix (MA) central globular domain ameliorated the NC15A assembly and processing defects, likely through enhancement of Gag multimerization and membrane binding capacities.

Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)

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

Hura, Greg L.; Menon, Angeli L.; Hammel, Michal

2009-07-20

We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes formore » 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.« less

Mono-Heteromeric Configurations of Gap Junction Channels Formed by Connexin43 and Connexin45 Reduce Unitary Conductance and Determine both Voltage Gating and Metabolic Flux Asymmetry

PubMed Central

Zhong, Guoqiang; Akoum, Nazem; Appadurai, Daniel A.; Hayrapetyan, Volodya; Ahmed, Osman; Martinez, Agustin D.; Beyer, Eric C.; Moreno, Alonso P.

2017-01-01

In cardiac tissues, the expression of multiple connexins (Cx40, Cx43, Cx45, and Cx30.2) is a requirement for proper development and function. Gap junctions formed by these connexins have distinct permeability and gating mechanisms. Since a single cell can express more than one connexin isoform, the formation of hetero-multimeric gap junction channels provides a tissue with an enormous repertoire of combinations to modulate intercellular communication. To study further the perm-selectivity and gating properties of channels containing Cx43 and Cx45, we studied two monoheteromeric combinations in which a HeLa cell co-transfected with Cx43 and Cx45 was paired with a cell expressing only one of these connexins. Macroscopic measurements of total conductance between cell pairs indicated a drastic reduction in total conductance for mono-heteromeric channels. In terms of Vj dependent gating, Cx43 homomeric connexons facing heteromeric connexons only responded weakly to voltage negativity. Cx45 homomeric connexons exhibited no change in Vj gating when facing heteromeric connexons. The distributions of unitary conductances (γj) for both mono-heteromeric channels were smaller than predicted, and both showed low permeability to the fluorescent dyes Lucifer yellow and Rhodamine123. For both mono-heteromeric channels, we observed flux asymmetry regardless of dye charge: flux was higher in the direction of the heteromeric connexon for MhetCx45 and in the direction of the homomeric Cx43 connexon for MhetCx43. Thus, our data suggest that co-expression of Cx45 and Cx43 induces the formation of heteromeric connexons with greatly reduced permeability and unitary conductance. Furthermore, it increases the asymmetry for voltage gating for opposing connexons, and it favors asymmetric flux of molecules across the junction that depends primarily on the size (not the charge) of the crossing molecules. PMID:28611680

Solution structure of the tandem acyl carrier protein domains from a polyunsaturated fatty acid synthase reveals beads-on-a-string configuration.

PubMed

Trujillo, Uldaeliz; Vázquez-Rosa, Edwin; Oyola-Robles, Delise; Stagg, Loren J; Vassallo, David A; Vega, Irving E; Arold, Stefan T; Baerga-Ortiz, Abel

2013-01-01

The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP domains for increasing the yield of fatty acids in bacterial cultures.

Bile Salt-Stimulated Lipase from Human Milk Binds DC-SIGN and Inhibits Human Immunodeficiency Virus Type 1 Transfer to CD4+ T Cells

PubMed Central

Naarding, Marloes A.; Dirac, Annette M.; Ludwig, Irene S.; Speijer, Dave; Lindquist, Susanne; Vestman, Eva-Lotta; Stax, Martijn J.; Geijtenbeek, Teunis B. H.; Pollakis, Georgios; Hernell, Olle; Paxton, William A.

2006-01-01

A wide range of pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, Mycobacterium, Leishmania, and Helicobacter pylori, can interact with dendritic cell (DC)-specific ICAM3-grabbing nonintegrin (DC-SIGN), expressed on DCs and a subset of B cells. More specifically, the interaction of the gp120 envelope protein of HIV-1 with DC-SIGN can facilitate the transfer of virus to CD4+ T lymphocytes in trans and enhance infection. We have previously demonstrated that a multimeric LeX component in human milk binds to DC-SIGN, preventing HIV-1 from interacting with this receptor. Biochemical analysis reveals that the compound is heat resistant, trypsin sensitive, and larger than 100 kDa, indicating a specific glycoprotein as the inhibitory compound. By testing human milk from three different mothers, we found the levels of DC-SIGN binding and viral inhibition to vary between samples. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and matrix-assisted laser desorption ionization analysis, we identified bile salt-stimulated lipase (BSSL), a Lewis X (LeX)-containing glycoprotein found in human milk, to be the major variant protein between the samples. BSSL isolated from human milk bound to DC-SIGN and inhibited the transfer of HIV-1 to CD4+ T lymphocytes. Two BSSL isoforms isolated from the same human milk sample showed differences in DC-SIGN binding, illustrating that alterations in the BSSL forms explain the differences observed. These results indicate that variations in BSSL lead to alterations in LeX expression by the protein, which subsequently alters the DC-SIGN binding capacity and the inhibitory effect on HIV-1 transfer. Identifying the specific molecular interaction between the different forms may aid in the future design of antimicrobial agents. PMID:17005819

Mono-Heteromeric Configurations of Gap Junction Channels Formed by Connexin43 and Connexin45 Reduce Unitary Conductance and Determine both Voltage Gating and Metabolic Flux Asymmetry.

PubMed

Zhong, Guoqiang; Akoum, Nazem; Appadurai, Daniel A; Hayrapetyan, Volodya; Ahmed, Osman; Martinez, Agustin D; Beyer, Eric C; Moreno, Alonso P

2017-01-01

In cardiac tissues, the expression of multiple connexins (Cx40, Cx43, Cx45, and Cx30.2) is a requirement for proper development and function. Gap junctions formed by these connexins have distinct permeability and gating mechanisms. Since a single cell can express more than one connexin isoform, the formation of hetero-multimeric gap junction channels provides a tissue with an enormous repertoire of combinations to modulate intercellular communication. To study further the perm-selectivity and gating properties of channels containing Cx43 and Cx45, we studied two monoheteromeric combinations in which a HeLa cell co-transfected with Cx43 and Cx45 was paired with a cell expressing only one of these connexins. Macroscopic measurements of total conductance between cell pairs indicated a drastic reduction in total conductance for mono-heteromeric channels. In terms of Vj dependent gating, Cx43 homomeric connexons facing heteromeric connexons only responded weakly to voltage negativity. Cx45 homomeric connexons exhibited no change in Vj gating when facing heteromeric connexons. The distributions of unitary conductances (γj) for both mono-heteromeric channels were smaller than predicted, and both showed low permeability to the fluorescent dyes Lucifer yellow and Rhodamine123. For both mono-heteromeric channels, we observed flux asymmetry regardless of dye charge: flux was higher in the direction of the heteromeric connexon for MhetCx45 and in the direction of the homomeric Cx43 connexon for MhetCx43. Thus, our data suggest that co-expression of Cx45 and Cx43 induces the formation of heteromeric connexons with greatly reduced permeability and unitary conductance. Furthermore, it increases the asymmetry for voltage gating for opposing connexons, and it favors asymmetric flux of molecules across the junction that depends primarily on the size (not the charge) of the crossing molecules.

Amyloid β oligomers induce interleukin-1β production in primary microglia in a cathepsin B- and reactive oxygen species-dependent manner

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

Taneo, Jun; Adachi, Takumi; Yoshida, Aiko

2015-03-13

Amyloid β (Aβ) peptide, a causative agent of Alzheimer's disease, forms two types of aggregates: oligomers and fibrils. These aggregates induce inflammatory responses, such as interleukin-1β (IL-1β) production by microglia, which are macrophage-like cells located in the brain. In this study, we examined the effect of the two forms of Aβ aggregates on IL-1β production in mouse primary microglia. We prepared Aβ oligomer and fibril from Aβ (1–42) peptide in vitro. We analyzed the characteristics of these oligomers and fibrils by electrophoresis and atomic force microscopy. Interestingly, Aβ oligomers but not Aβ monomers or fibrils induced robust IL-1β production in themore » presence of lipopolysaccharide. Moreover, Aβ oligomers induced endo/phagolysosome rupture, which released cathepsin B into the cytoplasm. Aβ oligomer-induced IL-1β production was inhibited not only by the cathepsin B inhibitor CA-074-Me but also by the reactive oxygen species (ROS) inhibitor N-acetylcysteine. Random chemical crosslinking abolished the ability of the oligomers to induce IL-1β. Thus, multimerization and fibrillization causes Aβ oligomers to lose the ability to induce IL-1β. These results indicate that Aβ oligomers, but not fibrils, induce IL-1β production in primary microglia in a cathepsin B- and ROS-dependent manner. - Highlights: • We prepared amyloid β (Aβ) fibrils with minimum contamination of Aβ oligomers. • Primary microglia (MG) produced IL-1β in response to Aβ oligomers, but not fibrils. • Only Aβ oligomers induced leakage of cathepsin B from endo/phagolysosomes. • IL-1β production in response to Aβ oligomers depended on both cathepsin B and ROS. • Crosslinking reduced the ability of the Aβ oligomers to induce IL-1β from MG.« less

Solution Structure of the Tandem Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase Reveals Beads-on-a-String Configuration

PubMed Central

Trujillo, Uldaeliz; Vázquez-Rosa, Edwin; Oyola-Robles, Delise; Stagg, Loren J.; Vassallo, David A.; Vega, Irving E.; Arold, Stefan T.; Baerga-Ortiz, Abel

2013-01-01

The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP domains for increasing the yield of fatty acids in bacterial cultures. PMID:23469090

Visual complexity: a review.

PubMed

Donderi, Don C

2006-01-01

The idea of visual complexity, the history of its measurement, and its implications for behavior are reviewed, starting with structuralism and Gestalt psychology at the beginning of the 20th century and ending with visual complexity theory, perceptual learning theory, and neural circuit theory at the beginning of the 21st. Evidence is drawn from research on single forms, form and texture arrays and visual displays. Form complexity and form probability are shown to be linked through their reciprocal relationship in complexity theory, which is in turn shown to be consistent with recent developments in perceptual learning and neural circuit theory. Directions for further research are suggested.

Priming by a novel universal influenza vaccine (Multimeric-001)-a gateway for improving immune response in the elderly population.

PubMed

Atsmon, Jacob; Caraco, Yoseph; Ziv-Sefer, Sagit; Shaikevich, Dimitry; Abramov, Ester; Volokhov, Inna; Bruzil, Svetlana; Haima, Kirsten Y; Gottlieb, Tanya; Ben-Yedidia, Tamar

2014-10-07

A new vaccine, "Multimeric-001" (M-001) has been recently developed, containing conserved, common linear influenza epitopes that activate both cellular and humoral arms of the immune system against a wide variety of influenza A and B strains. Apart from its direct action, M-001 is an attractive candidate for priming immune responses to seasonal influenza vaccine for the elderly population. The current clinical study was designed to assess M-001's standalone and priming action in participants over 65 years old. Evaluation of standalone action is based on induction of cell mediated immunity (CMI), since M-001 alone does not induce hemagglutinin inhibition (HAI) antibodies. This was a two-center, randomized, placebo-controlled study. 120 participants were randomized 1:1:1:1 into four groups to receive either two sequential non-adjuvanted or a single non-adjuvanted or a single adjuvanted intramuscular injection of 500 mcg M-001 (treatment), or one placebo (saline) injection, before receiving the trivalent inactivated influenza vaccine (TIV). Due to visual differences between placebo and treatment the study was partially blinded. HAI was evaluated at baseline and 3 weeks after standard TIV vaccination as a measure of M-001's efficacy. CMI responses were evaluated in a subset (10/group) of the participants. Participants were monitored for safety throughout the study. Overall the treatment was well-tolerated and safe, though sample sizes allowed only limited statistical analysis. M-001 priming resulted in enhanced seroconversion towards all three TIV strains, compared to priming with placebo. Significant elevation of influenza-specific CMI was observed following immunization with M-001 alone. The standalone and priming actions of M-001 were demonstrated in elderly participants despite the limitations of small population size and pre-existing HAI antibody titers in some participants. As a standalone vaccine, M-001 induced significant CMI to multiple strains and as a primer, M-001 enhanced HAI responses. Larger scale studies are warranted. NCT01419925. Copyright © 2014 Elsevier Ltd. All rights reserved.

Studies of molecular species of the human androgen receptor (AR): comparison of the physicochemical properties of the (/sup 3/H)methyltrienolone-AR complex formed in cytosol to the complex produced in intact genital skin fibroblasts

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

Keenan, B.S.; Greger, N.C.; Hedge, A.M.

1986-07-01

Two forms of the human genital skin fibroblast (GSF) androgen receptor (AR) complexed with (/sup 3/H)17 alpha-methyltrienolone were compared: 1) the intact complex formed in cytosol at 4 C (broken cell or B/C complex); and 2) the complex formed in the whole cell at 37 C (W/C complex). The intact form of the B/C complex was distinguished from partly degraded forms by the gel filtration profile in 0.5 M KCl. The W/C complex was considered to represent the transformed state of the receptor. The W/C complex had a smaller molecular radius than the B/C complex by gel filtration (Kav =more » 0.26-0.28 vs. 0.11-0.18). By low salt density gradient centrifugation, the B/C complex sedimented at 8.8S and the W/C complex at 6.6S. However, in 0.5 M KCl, each sedimented at 5.1S, and they were homogeneous, indicating that the monomeric forms differed markedly in molecular radius, but by only about 20,000 daltons in calculated mol wt (134,500 vs. 114,300 daltons). The complexes were separated from DNA, desalted, and compared by chromatography on DEAE-Sephacel and hydroxylapatite (HAP). The B/C complex bound readily to both column matrices and eluted from each as a sharp homogeneous peak: from DEAE at 172-190 mM KCl and from HAP at 123 mM phosphate. The W/C complex, however, was heterogeneous. One component did not bind to DEAE, and one eluted at 22-40 mM KCl. The W/C complex eluted from HAP as a peak at 42 mM, with a shoulder at 102 mM phosphate. Thus, transformation of the human genital skin fibroblast androgen receptor involves a major decrease in molecular radius and loss of negative charge with a possible loss of a 20,000-dalton macromolecular component.« less

21 CFR 520.1802 - Piperazine-carbon disulfide complex oral dosage forms.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Piperazine-carbon disulfide complex oral dosage forms. 520.1802 Section 520.1802 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... § 520.1802 Piperazine-carbon disulfide complex oral dosage forms. ...

Covariant symplectic structure of the complex Monge-Ampère equation

NASA Astrophysics Data System (ADS)

Nutku, Y.

2000-04-01

The complex Monge-Ampère equation is invariant under arbitrary holomorphic changes of the independent variables with unit Jacobian. We present its variational formulation where the action remains invariant under this infinite group. The new Lagrangian enables us to obtain the first symplectic 2-form for the complex Monge-Ampère equation in the framework of the covariant Witten-Zuckerman approach to symplectic structure. We base our considerations on a reformulation of the Witten-Zuckerman theory in terms of holomorphic differential forms. The first closed and conserved Witten-Zuckerman symplectic 2-form for the complex Monge-Ampère equation is obtained in arbitrary dimension and for all cases elliptic, hyperbolic and homogeneous. The connection of the complex Monge-Ampère equation with Ricci-flat Kähler geometry suggests the use of the Hilbert action principle as an alternative variational formulation. However, we point out that Hilbert's Lagrangian is a divergence for Kähler metrics and serves as a topological invariant rather than yielding the Euclideanized Einstein field equations. Nevertheless, since the Witten-Zuckerman theory employs only the boundary terms in the first variation of the action, Hilbert's Lagrangian can be used to obtain the second Witten-Zuckerman symplectic 2-form. This symplectic 2-form vanishes on shell, thus defining a Lagrangian submanifold. In its derivation the connection of the second symplectic 2-form with the complex Monge-Ampère equation is indirect but we show that it satisfies all the properties required of a symplectic 2-form for the complex elliptic, or hyperbolic Monge-Ampère equation when the dimension of the complex manifold is 3 or higher. The complex Monge-Ampère equation admits covariant bisymplectic structure for complex dimension 3, or higher. However, in the physically interesting case of n=2 we have only one symplectic 2-form. The extension of these results to the case of complex Monge-Ampère-Liouville equation is also presented.

RNA polymerases react differently at d(ApG) and d(GpG) adducts in DNA modified by cis-diamminedichloroplatinum(II)

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

Corda, Y.; Job, D.; Anin, M.F.

1992-02-25

Two duplexes (20-mers) were constructed containing either a single cis-(Pt(NH{sub 3}){sub 2}(d(GpG))) or cis-(Pt(NH{sub 3}){sub 2}(d(ApG))) intrastrand cross-link, the major DNA adducts of the antitumor drug cis-diamminedichloroplatinum(II). These synthetic duplexes were multimerized and the resultant polymers used as templates in single-step addition reactions of condensation of a single nucleoside triphosphate substrate to a dinucleotide primer (abortive elongation reaction) catalyzed by prokaryotic or eukaryotic RNA polymerases. Primer-substrate combinations were selected so as to direct trinucleotide product formation within the platinated bases of the templates. Transcription experiments established that cis-DDP-DNA adducts formed at d(ApG) or d(GpG) sites are not an absolute blockmore » to formation of a single phosphodiester bond by either Escherichia coli RNA polymerase or wheat germ RNA polymerase II. Furthermore, the kinetic data indicate that single-step addition reactions are much more impeded at the platinated d(GpG) than at the platinated d(ApG) site and that the mechanisms of inhibition of RNA polymerase activity are different at the two platinated sites. In particular, binding affinity between E. coli RNA polymerase and the d(GpG)-containing platinated template is lowered, as the apparent K{sub m} of enzyme for the platinated polymer is increased by a factor of 4-5. These results are discussed in reaction to the distortions induced in DNA by the two adducts.« less

Active suppression induced by repetitive self-epitopes protects against EAE development.

PubMed

Puentes, Fabiola; Dickhaut, Katharina; Hofstätter, Maria; Falk, Kirsten; Rötzschke, Olaf

2013-01-01

Autoimmune diseases result from a breakdown in self-tolerance to autoantigens. Self-tolerance is induced and sustained by central and peripheral mechanisms intended to deviate harmful immune responses and to maintain homeostasis, where regulatory T cells play a crucial role. The use of self-antigens in the study and treatment of a range of autoimmune diseases has been widely described; however, the mechanisms underlying the induced protection by these means are unclear. This study shows that protection of experimental autoimmune disease induced by T cell self-epitopes in a multimerized form (oligomers) is mediated by the induction of active suppression. The experimental autoimmune encephalomyelitis (EAE) animal model for multiple sclerosis was used to study the mechanisms of protection induced by the treatment of oligomerized T cell epitope of myelin proteolipid protein (PLP139-151). Disease protection attained by the administration of oligomers was shown to be antigen specific and effective in both prevention and treatment of ongoing EAE. Oligomer mediated tolerance was actively transferred by cells from treated mice into adoptive hosts. The induction of active suppression was correlated with the recruitment of cells in the periphery associated with increased production of IL-10 and reduction of the pro-inflammatory cytokine TNF-α. The role of suppressive cytokines was demonstrated by the reversion of oligomer-induced protection after in vivo blocking of either IL-10 or TGF-β cytokines. This study strongly supports an immunosuppressive role of repeat auto-antigens to control the development of EAE with potential applications in vaccination and antigen specific treatment of autoimmune diseases.

One-Step Chromatographic Purification of Helicobacter pylori Neutrophil-Activating Protein Expressed in Bacillus subtilis

PubMed Central

Shih, Kuo-Shun; Lin, Chih-Chang; Hung, Hsiao-Fang; Yang, Yu-Chi; Wang, Chung-An; Jeng, Kee-Ching; Fu, Hua-Wen

2013-01-01

Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection. PMID:23577158

Probing the structure, function, and interactions of the Escherichia coli H-NS and StpA proteins by using dominant negative derivatives.

PubMed

Williams, R M; Rimsky, S; Buc, H

1996-08-01

Twelve different dominant negative mutants of the Escherichia coli nucleoid-associated protein, H-NS, have been selected and characterized in vivo. The mutants are all severely defective in promoter repression activity in a strain lacking H-NS, and they all disrupt the repression normally exerted by H-NS at two of its target promoters. From the locations of the alterations in these mutants, which result in both large truncations and amino acid substitutions, we propose that H-NAS contains at least two distinct domains. The in vitro protein-protein cross-linking data presented in this report indicate that the proposed N-terminal domain of H-NS has a role in H-NS multimerization. StpA is a protein with known structural and functional homologies to H-NS. We have analyzed the extent of these homologies by constructing and studying StpA mutants predicted to be dominant negative. Our data indicate that the substitutions and deletions found in dominant negative H-NS have similar effects in the context of StpA. We conclude that the domain organizations and functions in StpA and H-NS are closely related. Furthermore, dominant negative H-NS can disrupt the activity of native StpA, and reciprocally, dominant negative StpA can disrupt the activity of native H-NS. We demonstrate that the N-terminal domain of H-NS can be chemically cross-linked to both full-length H-NS and StpA. We account for these observations by proposing that H-NS and StpA have the ability to form hybrid species.

The complete amino acid sequence of echinoidin, a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina. Homologies with mammalian and insect lectins.

PubMed

Giga, Y; Ikai, A; Takahashi, K

1987-05-05

The complete amino acid sequence of echinoidin, the proposed name for a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina, has been determined by sequencing the peptides obtained from tryptic, Staphylococcus aureus V8 protease, chymotryptic, and thermolysin digestions. Echinoidin is a multimeric protein (Giga, Y., Sutoh, K., and Ikai, A. (1985) Biochemistry 24, 4461-4467) whose subunit consists of a total of 147 amino acid residues and one carbohydrate chain attached to Ser38. The molecular weight of the polypeptide without carbohydrate was calculated to be 16,671. Each polypeptide chain contains seven half-cystines, and six of them form three disulfide bonds in the single polypeptide chain (Cys3-Cys14, Cys31-Cys141, and Cys116-Cys132), while Cys2 is involved in an interpolypeptide disulfide linkage. From secondary structure prediction by the method of Chou and Fasman (Chou, P. Y., and Fasman, G. D. (1974) Biochemistry 13, 211-222) the protein appears to be rich in beta-sheet and beta-turn structures and poor in alpha-helical structure. The sequence of the COOH-terminal half of echinoidin is highly homologous to those of the COOH-terminal carbohydrate recognition portions of rat liver mannose-binding protein and several other hepatic lectins. This COOH-terminal region of echinoidin is also homologous to the central portion of the lectin from the flesh fly Sarcophaga peregrina. Moreover, echinoidin contains an Arg-Gly-Asp sequence which has been proposed to be a basic functional unit in cellular recognition proteins.

The complex evolutionary history of big-eared horseshoe bats (Rhinolophus macrotis complex): insights from genetic, morphological and acoustic data

PubMed Central

Sun, Keping; Kimball, Rebecca T.; Liu, Tong; Wei, Xuewen; Jin, Longru; Jiang, Tinglei; Lin, Aiqing; Feng, Jiang

2016-01-01

Palaeoclimatic oscillations and different landscapes frequently result in complex population-level structure or the evolution of cryptic species. Elucidating the potential mechanisms is vital to understanding speciation events. However, such complex evolutionary patterns have rarely been reported in bats. In China, the Rhinolophus macrotis complex contains a large form and a small form, suggesting the existence of a cryptic bat species. Our field surveys found these two sibling species have a continuous and widespread distribution with partial sympatry. However, their evolutionary history has received little attention. Here, we used extensive sampling, morphological and acoustic data, as well as different genetic markers to investigate their evolutionary history. Genetic analyses revealed discordance between the mitochondrial and nuclear data. Mitochondrial data identified three reciprocally monophyletic lineages: one representing all small forms from Southwest China, and the other two containing all large forms from Central and Southeast China, respectively. The large form showed paraphyly with respect to the small form. However, clustering analyses of microsatellite and Chd1 gene sequences support two divergent clusters separating the large form and the small form. Moreover, morphological and acoustic analyses were consistent with nuclear data. This unusual pattern in the R. macrotis complex might be accounted for by palaeoclimatic oscillations, shared ancestral polymorphism and/or interspecific hybridization. PMID:27748429

Flotillin proteins recruit sphingosine to membranes and maintain cellular sphingosine-1-phosphate levels

PubMed Central

Riento, Kirsi; Zhang, Qifeng; Clark, Jonathan; Begum, Farida; Stephens, Elaine; Wakelam, Michael J.

2018-01-01

Sphingosine-1-phosphate (S1P) is an important lipid signalling molecule. S1P is produced via intracellular phosphorylation of sphingosine (Sph). As a lipid with a single fatty alkyl chain, Sph may diffuse rapidly between cellular membranes and through the aqueous phase. Here, we show that the absence of microdomains generated by multimeric assemblies of flotillin proteins results in reduced S1P levels. Cellular phenotypes of flotillin knockout mice, including changes in histone acetylation and expression of Isg15, are recapitulated when S1P synthesis is perturbed. Flotillins bind to Sph in vitro and increase recruitment of Sph to membranes in cells. Ectopic re-localisation of flotillins within the cell causes concomitant redistribution of Sph. The data suggest that flotillins may directly or indirectly regulate cellular sphingolipid distribution and signalling. PMID:29787576

Rubber pad forming - Efficient approach for the manufacturing of complex structured sheet metal blanks for food industry

NASA Astrophysics Data System (ADS)

Spoelstra, Paul; Djakow, Eugen; Homberg, Werner

2017-10-01

The production of complex organic shapes in sheet metals is gaining more importance in the food industry due to increasing functional and hygienic demands. Hence it is necessary to produce parts with complex geometries promoting cleanability and general sanitation leading to improvement of food safety. In this context, and especially when stainless steel has to be formed into highly complex geometries while maintaining desired surface properties, it is inevitable that alternative manufacturing processes will need to be used which meet these requirements. Rubber pad forming offers high potential when it comes to shaping complex parts with excellent surface quality, with virtually no tool marks and scratches. Especially in cases where only small series are to be produced, rubber pad forming processes offers both technological and economic advantages. Due to the flexible punch, variation in metal thickness can be used with the same forming tool. The investments to set-up Rubber pad forming is low in comparison to conventional sheet metal forming processes. The process facilitates production of shallow sheet metal parts with complex contours and bends. Different bending sequences in a multiple tool set-up can also be conducted. The planned contribution thus describes a brief overview of the rubber pad technology. It shows the prototype rubber pad forming machine which can be used to perform complex part geometries made from stainless steel (1.4301). Based on an analysis of the already existing systems and new machines for rubber pad forming processes, together with their process properties, influencing variables and areas of application, some relevant parts for the food industry are presented.

Neurophysiological evidence for whole form retrieval of complex derived words: a mismatch negativity study

PubMed Central

Hanna, Jeff; Pulvermüller, Friedemann

2014-01-01

Complex words can be seen as combinations of elementary units, decomposable into stems and affixes according to morphological rules. Alternatively, complex forms may be stored as single lexical entries and accessed as whole forms. This study uses an event-related potential brain response capable of indexing both whole-form retrieval and combinatorial processing, the Mismatch Negativity (MMN), to investigate early brain activity elicited by morphologically complex derived words in German. We presented complex words consisting of stems “sicher” (secure), or “sauber” (clean) combined with abstract nominalizing derivational affixes -heit or -keit, to form either congruent derived words: “Sicherheit” (security) and “Sauberkeit” (cleanliness), or incongruent derived pseudowords: *“Sicherkeit”, and *“Sauberheit”. Using this orthogonal design, it was possible to record brain responses for -heit and -keit in both congruent and incongruent contexts, therefore balancing acoustic variance. Previous research has shown that incongruent combinations of symbols elicit a stronger MMN than congruent combinations, but that single words or constructions stored as whole forms elicit a stronger MMN than pseudowords or non-existent constructions. We found that congruent derived words elicited a stronger MMN than incongruent derived words, beginning about 150 ms after perception of the critical morpheme. This pattern of results is consistent with whole-form storage of morphologically complex derived words as lexical units, or mini-constructions. Using distributed source localization methods, the MMN enhancement for well-formed derivationally complex words appeared to be most prominent in the left inferior anterior-temporal, bilateral superior parietal and bilateral post-central, supra-marginal areas. In addition, neurophysiological results reflected the frequency of derived forms, thus providing further converging evidence for whole form storage and against a combinatorial mechanism. PMID:25414658

Neurophysiological evidence for whole form retrieval of complex derived words: a mismatch negativity study.

PubMed

Hanna, Jeff; Pulvermüller, Friedemann

2014-01-01

Complex words can be seen as combinations of elementary units, decomposable into stems and affixes according to morphological rules. Alternatively, complex forms may be stored as single lexical entries and accessed as whole forms. This study uses an event-related potential brain response capable of indexing both whole-form retrieval and combinatorial processing, the Mismatch Negativity (MMN), to investigate early brain activity elicited by morphologically complex derived words in German. We presented complex words consisting of stems "sicher" (secure), or "sauber" (clean) combined with abstract nominalizing derivational affixes -heit or -keit, to form either congruent derived words: "Sicherheit" (security) and "Sauberkeit" (cleanliness), or incongruent derived pseudowords: *"Sicherkeit", and *"Sauberheit". Using this orthogonal design, it was possible to record brain responses for -heit and -keit in both congruent and incongruent contexts, therefore balancing acoustic variance. Previous research has shown that incongruent combinations of symbols elicit a stronger MMN than congruent combinations, but that single words or constructions stored as whole forms elicit a stronger MMN than pseudowords or non-existent constructions. We found that congruent derived words elicited a stronger MMN than incongruent derived words, beginning about 150 ms after perception of the critical morpheme. This pattern of results is consistent with whole-form storage of morphologically complex derived words as lexical units, or mini-constructions. Using distributed source localization methods, the MMN enhancement for well-formed derivationally complex words appeared to be most prominent in the left inferior anterior-temporal, bilateral superior parietal and bilateral post-central, supra-marginal areas. In addition, neurophysiological results reflected the frequency of derived forms, thus providing further converging evidence for whole form storage and against a combinatorial mechanism.

Dose response relationship in anti-stress gene regulatory networks.

PubMed

Zhang, Qiang; Andersen, Melvin E

2007-03-02

To maintain a stable intracellular environment, cells utilize complex and specialized defense systems against a variety of external perturbations, such as electrophilic stress, heat shock, and hypoxia, etc. Irrespective of the type of stress, many adaptive mechanisms contributing to cellular homeostasis appear to operate through gene regulatory networks that are organized into negative feedback loops. In general, the degree of deviation of the controlled variables, such as electrophiles, misfolded proteins, and O2, is first detected by specialized sensor molecules, then the signal is transduced to specific transcription factors. Transcription factors can regulate the expression of a suite of anti-stress genes, many of which encode enzymes functioning to counteract the perturbed variables. The objective of this study was to explore, using control theory and computational approaches, the theoretical basis that underlies the steady-state dose response relationship between cellular stressors and intracellular biochemical species (controlled variables, transcription factors, and gene products) in these gene regulatory networks. Our work indicated that the shape of dose response curves (linear, superlinear, or sublinear) depends on changes in the specific values of local response coefficients (gains) distributed in the feedback loop. Multimerization of anti-stress enzymes and transcription factors into homodimers, homotrimers, or even higher-order multimers, play a significant role in maintaining robust homeostasis. Moreover, our simulation noted that dose response curves for the controlled variables can transition sequentially through four distinct phases as stressor level increases: initial superlinear with lesser control, superlinear more highly controlled, linear uncontrolled, and sublinear catastrophic. Each phase relies on specific gain-changing events that come into play as stressor level increases. The low-dose region is intrinsically nonlinear, and depending on the level of local gains, presence of gain-changing events, and degree of feedforward gene activation, this region can appear as superlinear, sublinear, or even J-shaped. The general dose response transition proposed here was further examined in a complex anti-electrophilic stress pathway, which involves multiple genes, enzymes, and metabolic reactions. This work would help biologists and especially toxicologists to better assess and predict the cellular impact brought about by biological stressors.

Structure of the active form of human origin recognition complex and its ATPase motor module

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

Tocilj, Ante; On, Kin Fan; Yuan, Zuanning

Binding of the Origin Recognition Complex (ORC) to origins of replication marks the first step in the initiation of replication of the genome in all eukaryotic cells. Here, we report the structure of the active form of human ORC determined by X-ray crystallography and cryo-electron microscopy. The complex is composed of an ORC1/4/5 motor module lobe in an organization reminiscent of the DNA polymerase clamp loader complexes. A second lobe contains the ORC2/3 subunits. The complex is organized as a double-layered shallow corkscrew, with the AAA+ and AAA+-like domains forming one layer, and the winged-helix domains (WHDs) forming a topmore » layer. CDC6 fits easily between ORC1 and ORC2, completing the ring and the DNA-binding channel, forming an additional ATP hydrolysis site. Analysis of the ATPase activity of the complex provides a basis for understanding ORC activity as well as molecular defects observed in Meier-Gorlin Syndrome mutations.« less

Proteome analysis of the plant pathogen Xylella fastidiosa reveals major cellular and extracellular proteins and a peculiar codon bias distribution.

PubMed

Smolka, Marcus Bustamante; Martins-de-Souza, Daniel; Martins, Daniel; Winck, Flavia Vischi; Santoro, Carlos Eduardo; Castellari, Rafael Ramos; Ferrari, Fernanda; Brum, Itaraju Junior; Galembeck, Eduardo; Della Coletta Filho, Helvécio; Machado, Marcos Antonio; Marangoni, Sergio; Novello, Jose Camillo

2003-02-01

The bacteria Xylella fastidiosa is the causative agent of a number of economically important crop diseases, including citrus variegated chlorosis. Although its complete genome is already sequenced, X. fastidiosa is very poorly characterized by biochemical approaches at the protein level. In an initial effort to characterize protein expression in X. fastidiosa we used one- and two-dimensional gel electrophoresis and mass spectrometry to identify the products of 142 genes present in a whole cell extract and in an extracellular fraction of the citrus isolated strain 9a5c. Of particular interest for the study of pathogenesis are adhesion and secreted proteins. Homologs to proteins from three different adhesion systems (type IV fimbriae, mrk pili and hsf surface fibrils) were found to be coexpressed, the last two being detected only as multimeric complexes in the high molecular weight region of one-dimensional electrophoresis gels. Using a procedure to extract secreted proteins as well as proteins weakly attached to the cell surface we identified 30 different proteins including toxins, adhesion related proteins, antioxidant enzymes, different types of proteases and 16 hypothetical proteins. These data suggest that the intercellular space of X. fastidiosa colonies is a multifunctional microenvironment containing proteins related to in vivo bacterial survival and pathogenesis. A codon usage analysis of the most expressed proteins from the whole cell extract revealed a low biased distribution, which we propose is related to the slow growing nature of X. fastidiosa. A database of the X. fastidiosa proteome was developed and can be accessed via the internet (URL: www.proteome.ibi.unicamp.br).

A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

PubMed Central

Rodermel, S; Haley, J; Jiang, C Z; Tsai, C H; Bogorad, L

1996-01-01

Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6 Fig. 7 Fig. 8 PMID:8632983

Affinity purification of angiotensin type 2 receptors from N1E-115 cells: evidence for agonist-induced formation of multimeric complexes.

PubMed

Siemens, I R; Yee, D K; Reagan, L P; Fluharty, S J

1994-01-01

The murine neuroblastoma N1E-115 cell line possesses type 1 and type 2 angiotensin II (AngII) receptor subtypes. In vitro differentiation of these cells substantially increases the density of the AT2-receptor subtype, whereas the density of the AT1 receptors remains unchanged. In the present study, we report that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) selectively solubilized AT2 receptors from N1E-115 cell membranes and that these receptors could be purified further to near homogeneity by affinity chromatography. More specifically, the presence of an agonist (AngII) during affinity purification of AT2 receptors resulted in the elution of high (110-kDa) and low (66-kDa) molecular mass proteins as determined by gel electrophoresis under nonreducing conditions. In contrast, when the nonselective antagonist Sar1,Ile8-AngII was used during purification, only the lower 66-kDa protein was observed. Affinity purification in the presence of the peptide and nonpeptide AT2-receptor antagonists CGP42112A and PD123319 also resulted in elution of the same 66-kDa protein, but unlike that in the presence of Sar1,Ile8-AngII, some of the high molecular weight site was observed as well. On the other hand, Losartan, an AT1-receptor antagonist, was completely ineffective in eluting any AngII receptors from the affinity column, further confirming their AT2 identity. After agonist elution, the 110-kDa band dissociated into two low molecular mass bands of 66 kDa and 54 kDa when sodium dodecyl sulfate-gel electrophoresis was run under reducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of factor VIII pharmaceutical preparations by means of MudPIT proteomic approach.

PubMed

Basilico, Fabrizio; Nardini, Ilaria; Mori, Filippo; Brambilla, Elena; Benazzi, Louise; De Palma, Antonella; Rosti, Enrico; Farina, Claudio; Mauri, PierLuigi

2010-09-21

For a good clinical outcome of Haemophilia A substitutive therapy a detailed characterization of factor VIII (FVIII) concentrates is required, in order to disclose the eventual relations between differently composed concentrates and their biological effects. This preliminary work could be a first step towards a deep structural characterization of FVIII concentrates, using the fast and simply manageable MudPIT technology, which enables the identification and characterization of protein mixtures taking advantage of both the high separation capacity of two-dimensional chromatography and the powerful peptide characterization ability of tandem mass spectrometry. The aim of this study was to evaluate the suitability of for the characterization of FVIII molecule in complex mixtures such its commercial concentrates, both plasma-derived and recombinant, and for the determination of the protein composition of different FVIII preparations. By means of Multidimensional Protein Identification Technology (MudPIT) it was possible to assess the presence of factor VIII in its preparations and to identify most of the contaminant proteins without gel separation. In particular, 125 and 42 proteins were identified in plasma-derived and recombinant concentrates, respectively. Concerning investigation of FVIII, 24 different peptides were identified in plasma-derived corresponding to 7, 29, 27, 19 and 67 of percentage coverage for A1, A2, A3, C1 and C2 domains, respectively. About its multimeric carrier von Willebrand factor (VWF), we have sequenced 42% of domain interacting with A3 and C2 domains of FVIII. Finally, it has been observed that normalized parameters, such as total peptide hits obtained by SEQUEST may be used for evaluation of the relative abundance of FVIII in different preparations. Copyright 2010 Elsevier B.V. All rights reserved.

Unification of [FeFe]-hydrogenases into three structural and functional groups.

PubMed

Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R; Refai, Mohammed; Schut, Gerrit J; King, Paul W; Maness, Pin-Ching; Adams, Michael W W; Peters, John W; Bothner, Brian; Boyd, Eric S

2016-09-01

[FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H2). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H2. To identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA, and regulatory proteins encoded in HydA gene neighborhoods. HydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggest that they are post-translationally modified by phosphorylation. These results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA. This classification scheme provides a framework for future biochemical and mutagenesis studies to elucidate the functional role of Hyd enzymes. Copyright © 2016 Elsevier B.V. All rights reserved.

PUF60 variants cause a syndrome of ID, short stature, microcephaly, coloboma, craniofacial, cardiac, renal and spinal features

PubMed Central

Low, Karen J; Ansari, Morad; Abou Jamra, Rami; Clarke, Angus; El Chehadeh, Salima; FitzPatrick, David R; Greenslade, Mark; Henderson, Alex; Hurst, Jane; Keller, Kory; Kuentz, Paul; Prescott, Trine; Roessler, Franziska; Selmer, Kaja K; Schneider, Michael C; Stewart, Fiona; Tatton-Brown, Katrina; Thevenon, Julien; Vigeland, Magnus D; Vogt, Julie; Willems, Marjolaine; Zonana, Jonathan; Study, D D D; Smithson, Sarah F

2017-01-01

PUF60 encodes a nucleic acid-binding protein, a component of multimeric complexes regulating RNA splicing and transcription. In 2013, patients with microdeletions of chromosome 8q24.3 including PUF60 were found to have developmental delay, microcephaly, craniofacial, renal and cardiac defects. Very similar phenotypes have been described in six patients with variants in PUF60, suggesting that it underlies the syndrome. We report 12 additional patients with PUF60 variants who were ascertained using exome sequencing: six through the Deciphering Developmental Disorders Study and six through similar projects. Detailed phenotypic analysis of all patients was undertaken. All 12 patients had de novo heterozygous PUF60 variants on exome analysis, each confirmed by Sanger sequencing: four frameshift variants resulting in premature stop codons, three missense variants that clustered within the RNA recognition motif of PUF60 and five essential splice-site (ESS) variant. Analysis of cDNA from a fibroblast cell line derived from one of the patients with an ESS variants revealed aberrant splicing. The consistent feature was developmental delay and most patients had short stature. The phenotypic variability was striking; however, we observed similarities including spinal segmentation anomalies, congenital heart disease, ocular colobomata, hand anomalies and (in two patients) unilateral renal agenesis/horseshoe kidney. Characteristic facial features included micrognathia, a thin upper lip and long philtrum, narrow almond-shaped palpebral fissures, synophrys, flared eyebrows and facial hypertrichosis. Heterozygote loss-of-function variants in PUF60 cause a phenotype comprising growth/developmental delay and craniofacial, cardiac, renal, ocular and spinal anomalies, adding to disorders of human development resulting from aberrant RNA processing/spliceosomal function. PMID:28327570

Binding of Y-P30 to Syndecan 2/3 Regulates the Nuclear Localization of CASK

PubMed Central

Landgraf, Peter; Mikhaylova, Marina; Macharadze, Tamar; Borutzki, Corinna; Zenclussen, Ana-Claudia; Wahle, Petra; Kreutz, Michael R.

2014-01-01

The survival promoting peptide Y-P30 has documented neuroprotective effects as well as cell survival and neurite outgrowth promoting activity in vitro and in vivo. Previous work has shown that multimerization of the peptide with pleiotrophin (PTN) and subsequent binding to syndecan (SDC) -2 and -3 is involved in its neuritogenic effects. In this study we show that Y-P30 application regulates the nuclear localization of the SDC binding partner Calcium/calmodulin-dependent serine kinase (CASK) in neuronal primary cultures during development. In early development at day in vitro (DIV) 8 when mainly SDC-3 is expressed supplementation of the culture medium with Y-P30 reduces nuclear CASK levels whereas it has the opposite effect at DIV 18 when SDC-2 is the dominant isoform. In the nucleus CASK regulates gene expression via its association with the T-box transcription factor T-brain-1 (Tbr-1) and we indeed found that gene expression of downstream targets of this complex, like the GluN2B NMDA-receptor, exhibits a corresponding down- or up-regulation at the mRNA level. The differential effect of Y-P30 on the nuclear localization of CASK correlates with its ability to induce shedding of the ectodomain of SDC-2 but not -3. shRNA knockdown of SDC-2 at DIV 18 and SDC-3 at DIV 8 completely abolished the effect of Y-P30 supplementation on nuclear CASK levels. During early development a protein knockdown of SDC-3 also attenuated the effect of Y-P30 on axon outgrowth. Taken together these data suggest that Y-P30 can control the nuclear localization of CASK in a SDC-dependent manner. PMID:24498267

Unification of [FeFe]-hydrogenases into three structural and functional groups

DOE PAGES

Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R.; ...

2016-05-27

[FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H 2). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H 2. To identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA,more » and regulatory proteins encoded in HydA gene neighborhoods. HydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggests that they are post-translationally modified by phosphorylation. In conclusion, these results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA.« less

Experimental verification of the kinetic theory of FRET using optical microspectroscopy and obligate oligomers.

PubMed

Patowary, Suparna; Pisterzi, Luca F; Biener, Gabriel; Holz, Jessica D; Oliver, Julie A; Wells, James W; Raicu, Valerică

2015-04-07

Förster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy from an optically excited donor molecule (D) to an acceptor molecule (A) in the ground state. The underlying theory predicting the dependence of the FRET efficiency on the sixth power of the distance between D and A has stood the test of time. In contrast, a comprehensive kinetic-based theory developed recently for FRET efficiencies among multiple donors and acceptors in multimeric arrays has waited for further testing. That theory has been tested in the work described in this article using linked fluorescent proteins located in the cytoplasm and at the plasma membrane of living cells. The cytoplasmic constructs were fused combinations of Cerulean as donor (D), Venus as acceptor (A), and a photo-insensitive molecule (Amber) as a nonfluorescent (N) place holder: namely, NDAN, NDNA, and ADNN duplexes, and the fully fluorescent quadruplex ADAA. The membrane-bound constructs were fused combinations of GFP2 as donor (D) and eYFP as acceptor (A): namely, two fluorescent duplexes (i.e., DA and AD) and a fluorescent triplex (ADA). According to the theory, the FRET efficiency of a multiplex such as ADAA or ADA can be predicted from that of analogs containing a single acceptor (e.g., NDAN, NDNA, and ADNN, or DA and AD, respectively). Relatively small but statistically significant differences were observed between the measured and predicted FRET efficiencies of the two multiplexes. While elucidation of the cause of this mismatch could be a worthy endeavor, the discrepancy does not appear to question the theoretical underpinnings of a large family of FRET-based methods for determining the stoichiometry and quaternary structure of complexes of macromolecules in living cells. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment

PubMed Central

Iezzi, María Elena; Policastro, Lucía; Werbajh, Santiago; Podhajcer, Osvaldo; Canziani, Gabriela Alicia

2018-01-01

Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs), in particular those engineered from the variable heavy-chain fragment (VHH gene) found in Camelidae heavy-chain antibodies (or IgG2 and IgG3), are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR) fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition “modules” to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo. Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads. PMID:29520274

Content of mitochondrial calcium uniporter (MCU) in cardiomyocytes is regulated by microRNA-1 in physiologic and pathologic hypertrophy

PubMed Central

Zaglia, Tania; Ceriotti, Paola; Campo, Antonio; Borile, Giulia; Armani, Andrea; Carullo, Pierluigi; Prando, Valentina; Coppini, Raffaele; Vida, Vladimiro; Stølen, Tomas O.; Ulrik, Wisløff; Cerbai, Elisabetta; Stellin, Giovanni; Faggian, Giuseppe; De Stefani, Diego; Sandri, Marco; Rizzuto, Rosario; Di Lisa, Fabio; Pozzan, Tullio; Catalucci, Daniele; Mongillo, Marco

2017-01-01

The mitochondrial Ca2+ uniporter complex (MCUC) is a multimeric ion channel which, by tuning Ca2+ influx into the mitochondrial matrix, finely regulates metabolic energy production. In the heart, this dynamic control of mitochondrial Ca2+ uptake is fundamental for cardiomyocytes to adapt to either physiologic or pathologic stresses. Mitochondrial calcium uniporter (MCU), which is the core channel subunit of MCUC, has been shown to play a critical role in the response to β-adrenoreceptor stimulation occurring during acute exercise. The molecular mechanisms underlying the regulation of MCU, in conditions requiring chronic increase in energy production, such as physiologic or pathologic cardiac growth, remain elusive. Here, we show that microRNA-1 (miR-1), a member of the muscle-specific microRNA (myomiR) family, is responsible for direct and selective targeting of MCU and inhibition of its translation, thereby affecting the capacity of the mitochondrial Ca2+ uptake machinery. Consistent with the role of miR-1 in heart development and cardiomyocyte hypertrophic remodeling, we additionally found that MCU levels are inversely related with the myomiR content, in murine and, remarkably, human hearts from both physiologic (i.e., postnatal development and exercise) and pathologic (i.e., pressure overload) myocardial hypertrophy. Interestingly, the persistent activation of β-adrenoreceptors is likely one of the upstream repressors of miR-1 as treatment with β-blockers in pressure-overloaded mouse hearts prevented its down-regulation and the consequent increase in MCU content. Altogether, these findings identify the miR-1/MCU axis as a factor in the dynamic adaptation of cardiac cells to hypertrophy. PMID:29073097

Development of second generation peptides modulating cellular adiponectin receptor responses

NASA Astrophysics Data System (ADS)

Otvos, Laszlo; Knappe, Daniel; Hoffmann, Ralf; Kovalszky, Ilona; Olah, Julia; Hewitson, Tim; Stawikowska, Roma; Stawikowski, Maciej; Cudic, Predrag; Lin, Feng; Wade, John; Surmacz, Eva; Lovas, Sandor

2014-10-01

The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC). In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML) cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399). The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM - low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400) was designed that counteracted 10-1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400) at similar concentrations will be an important target validation tool to study adiponectin functions.

Expression, purification and preliminary biochemical and structural characterization of the leucine rich repeat namesake domain of leucine rich repeat kinase 2.

PubMed

Vancraenenbroeck, Renée; Lobbestael, Evy; Weeks, Stephen D; Strelkov, Sergei V; Baekelandt, Veerle; Taymans, Jean-Marc; De Maeyer, Marc

2012-03-01

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, Ras of complex proteins) and kinase domains and a connecting COR (C-terminus of ROC) domain. In contrast, the precise functions of the protein-protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR(LRRK2)) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR(LRRK2) in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR(LRRK2) thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% α-helices and 21% β-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR(LRRK2) multimerization was detected via cross-linking studies. Finally, the LRR(LRRK2) clinical mutations did not influence LRR(LRRK2) secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR(LRRK2) inter- and intramolecular interactions. Copyright © 2011 Elsevier B.V. All rights reserved.

High-mobility group 1/2 proteins are essential for initiating rolling-circle-type DNA replication at a parvovirus hairpin origin.

PubMed

Cotmore, S F; Tattersall, P

1998-11-01

Rolling-circle replication is initiated by a replicon-encoded endonuclease which introduces a single-strand nick into specific origin sequences, becoming covalently attached to the 5' end of the DNA at the nick and providing a 3' hydroxyl to prime unidirectional, leading-strand synthesis. Parvoviruses, such as minute virus of mice (MVM), have adapted this mechanism to amplify their linear single-stranded genomes by using hairpin telomeres which sequentially unfold and refold to shuttle the replication fork back and forth along the genome, creating a continuous, multimeric DNA strand. The viral initiator protein, NS1, then excises individual genomes from this continuum by nicking and reinitiating synthesis at specific origins present within the hairpin sequences. Using in vitro assays to study ATP-dependent initiation within the right-hand (5') MVM hairpin, we have characterized a HeLa cell factor which is absolutely required to allow NS1 to nick this origin. Unlike parvovirus initiation factor (PIF), the cellular complex which activates NS1 endonuclease activity at the left-hand (3') viral origin, the host factor which activates the right-hand hairpin elutes from phosphocellulose in high salt, has a molecular mass of around 25 kDa, and appears to bind preferentially to structured DNA, suggesting that it might be a member of the high-mobility group 1/2 (HMG1/2) protein family. This prediction was confirmed by showing that purified calf thymus HMG1 and recombinant human HMG1 or murine HMG2 could each substitute for the HeLa factor, activating the NS1 endonuclease in an origin-specific nicking reaction.

Computational analysis of an autophagy/translation switch based on mutual inhibition of MTORC1 and ULK1

DOE PAGES

Szymańska, Paulina; Martin, Katie R.; MacKeigan, Jeffrey P.; ...

2015-03-11

We constructed a mechanistic, computational model for regulation of (macro)autophagy and protein synthesis (at the level of translation). The model was formulated to study the system-level consequences of interactions among the following proteins: two key components of MTOR complex 1 (MTORC1), namely the protein kinase MTOR (mechanistic target of rapamycin) and the scaffold protein RPTOR; the autophagy-initiating protein kinase ULK1; and the multimeric energy-sensing AMP-activated protein kinase (AMPK). Inputs of the model include intrinsic AMPK kinase activity, which is taken as an adjustable surrogate parameter for cellular energy level or AMP:ATP ratio, and rapamycin dose, which controls MTORC1 activity. Outputsmore » of the model include the phosphorylation level of the translational repressor EIF4EBP1, a substrate of MTORC1, and the phosphorylation level of AMBRA1 (activating molecule in BECN1-regulated autophagy), a substrate of ULK1 critical for autophagosome formation. The model incorporates reciprocal regulation of mTORC1 and ULK1 by AMPK, mutual inhibition of MTORC1 and ULK1, and ULK1-mediated negative feedback regulation of AMPK. Through analysis of the model, we find that these processes may be responsible, depending on conditions, for graded responses to stress inputs, for bistable switching between autophagy and protein synthesis, or relaxation oscillations, comprising alternating periods of autophagy and protein synthesis. A sensitivity analysis indicates that the prediction of oscillatory behavior is robust to changes of the parameter values of the model. The model provides testable predictions about the behavior of the AMPK-MTORC1-ULK1 network, which plays a central role in maintaining cellular energy and nutrient homeostasis.« less

Trapping Mode Dipolar DC Collisional Activation in the RF-Only Ion Guide of a Linear Ion Trap/Time-of-Flight Instrument for Gaseous Bio-Ion Declustering

PubMed Central

Webb, Ian K.; Gao, Yang; Londry, Frank A.; McLuckey, Scott A.

2013-01-01

The application of dipolar DC (DDC) to the RF-only ion guide (Q0) of a hybrid quadrupole/time-of-flight (QqTOF) mass spectrometer for collision-induced declustering of large bio-ions is described. As a broadband technique, ion trap DDC collision activation (CA) is employed to decluster ions simultaneously over a relatively broad mass-to-charge range. Declustering DDC CA can yield significantly narrower peaks relative to those observed in the absence of declustering methods, depending upon the extent of non-covalent adduction associated with the ions, and can also be used in conjunction with other methods, such as nozzle-skimmer collisional activation. The key experimental variables in the DDC experiment are the dipolar DC voltage (VDDC), VRF, and the time over which VDDC is applied. The VDDC/VRF ratio is key to the extent to which ion temperatures are elevated and also influences the upper mass-to-charge limit for ion storage. The VDDC/VRF ratio affects ion temperatures and upper m/z limit in opposing directions. That is, as the ratio increases, ion temperature increases whereas the upper m/z storage limit decreases. However, for a given VDDC/VRF ratio, the upper m/z storage limit can be increased by increasing VRF, at the expense of the lower m/z limit for ion storage. The key value of the approach is that it affords a relatively precise degree of control over ion temperatures as well as the time over which they are elevated to the higher temperature. The utility of the method is illustrated by the application of ion trap DDC CA in Q0 to oligonucleotide, protein, and multimeric protein complex analyte ions. PMID:24078247

Zea mays iRS1563: A Comprehensive Genome-Scale Metabolic Reconstruction of Maize Metabolism

PubMed Central

Saha, Rajib; Suthers, Patrick F.; Maranas, Costas D.

2011-01-01

The scope and breadth of genome-scale metabolic reconstructions have continued to expand over the last decade. Herein, we introduce a genome-scale model for a plant with direct applications to food and bioenergy production (i.e., maize). Maize annotation is still underway, which introduces significant challenges in the association of metabolic functions to genes. The developed model is designed to meet rigorous standards on gene-protein-reaction (GPR) associations, elementally and charged balanced reactions and a biomass reaction abstracting the relative contribution of all biomass constituents. The metabolic network contains 1,563 genes and 1,825 metabolites involved in 1,985 reactions from primary and secondary maize metabolism. For approximately 42% of the reactions direct literature evidence for the participation of the reaction in maize was found. As many as 445 reactions and 369 metabolites are unique to the maize model compared to the AraGEM model for A. thaliana. 674 metabolites and 893 reactions are present in Zea mays iRS1563 that are not accounted for in maize C4GEM. All reactions are elementally and charged balanced and localized into six different compartments (i.e., cytoplasm, mitochondrion, plastid, peroxisome, vacuole and extracellular). GPR associations are also established based on the functional annotation information and homology prediction accounting for monofunctional, multifunctional and multimeric proteins, isozymes and protein complexes. We describe results from performing flux balance analysis under different physiological conditions, (i.e., photosynthesis, photorespiration and respiration) of a C4 plant and also explore model predictions against experimental observations for two naturally occurring mutants (i.e., bm1 and bm3). The developed model corresponds to the largest and more complete to-date effort at cataloguing metabolism for a plant species. PMID:21755001

Identification of the hydrophobic strand in the A–B loop of leptin as major binding site III: implications for large-scale preparation of potent recombinant human and ovine leptin antagonists

PubMed Central

Niv-Spector, Leonora; Gonen-Berger, Dana; Gourdou, Isabelle; Biener, Eva; Gussakovsky, Eugene E.; Benomar, Yackir; Ramanujan, Krishnan V.; Taouis, Mohammed; Herman, Brian; Callebaut, Isabelle; Djiane, Jean; Gertler, Arieh

2005-01-01

Interaction of leptin with its receptors resembles that of interleukin-6 and granulocyte colony-stimulating factor, which interact with their receptors through binding sites I–III. Site III plays a pivotal role in receptors' dimerization or tetramerization and subsequent activation. Leptin's site III also mediates the formation of an active multimeric complex through its interaction with the IGD (immunoglobulin-like domain) of LEPRs (leptin receptors). Using a sensitive hydrophobic cluster analysis of leptin's and LEPR's sequences, we identified hydrophobic stretches in leptin's A–B loop (amino acids 39–42) and in the N-terminal end of LEPR's IGD (amino acids 325–328) that are predicted to participate in site III and to interact with each other in a β-sheet-like configuration. To verify this hypothesis, we prepared and purified to homogeneity (as verified by SDS/PAGE, gel filtration and reverse-phase chromatography) several alanine muteins of amino acids 39–42 in human and ovine leptins. CD analyses revealed that those mutations hardly affect the secondary structure. All muteins acted as true antagonists, i.e. they bound LEPR with an affinity similar to the wild-type hormone, had no agonistic activity and specifically inhibited leptin action in several leptin-responsive in vitro bioassays. Alanine mutagenesis of LEPR's IGD (amino acids 325–328) drastically reduced its biological but not binding activity, indicating the importance of this region for interaction with leptin's site III. FRET (fluorescence resonance energy transfer) microscopy experiments have documented that the transient FRET signalling occurring upon exposure to leptin results not from binding of the ligand, but from ligand-induced oligomerization of LEPRs mediated by leptin's site III. PMID:15952938

Generation kinetics of boron-oxygen complexes in p-type compensated c-Si

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

Wu, Yichao; Yu, Xuegong, E-mail: yuxuegong@zju.edu.cn; Chen, Peng

2014-03-10

Kinetics characteristics of boron-oxygen complexes responsible for light-induced degradation in p-type compensated c-Si have been investigated. The generation of B-O complexes is well fitted by a fast-forming process and a slow-forming one. Activation energies of complexes generation during the fast-forming process are determined to be 0.29 and 0.24 eV in compensated and non-compensated c-Si, respectively, and those during the slow-forming process are the same, about 0.44 eV. Moreover, it is found that the pre-exponential factors of complexes generation in compensated c-Si is proportional to the square of the net doping concentration, which suggests that the latent centers should exist.

Solubility and transdermal permeation properties of a dehydroepiandrosterone cyclodextrin complex from hydrophilic and lipophilic vehicles.

PubMed

Ceschel, GianCarlo; Bergamante, Valentina; Maffei, Paola; Lombardi Borgia, Simone; Calabrese, Valeria; Biserni, Stefano; Ronchi, Celestino

2005-01-01

The permeation ability of a compound is due principally to its concentration in the vehicle and to its aptitude to cross the stratum corneum of the skin. In this work ex-vivo permeation studies on newly developed formulations containing dehydroepiandrosterone (DHEA) were carried out to investigate vehicles that increase drug permeation through the skin. To enhance the solubility of DHEA, its complex form with alpha-cyclodextrin was used. In addition, the two forms (pure drug and complex form) were introduced in hydrophilic (water), lipophilic (paraffin oil), and microemulsion vehicles to evaluate the synergic effect of cyclodextrins and microemulsion vehicles on solubility and permeation. From the results, DHEA solubility is notably conditioned by the type of the vehicle used: the highest solubilities (both for pure and complex drug forms) were obtained with microemulsion, followed by paraffin oil and water. Moreover, in all the studied vehicles, the c-DHEA was more soluble than DHEA. Permeation profile fluxes showed very interesting differences. That reflect the varying drug forms (pure drug and complex form), vehicles used, and drug concentrations in the vehicles. The major flux was obtained in complex of DHEA with alpha-cyclodextrins in the microemulsion vehicle. Therefore, this type of vehicle and drug form would be very useful in the development of a topical formulation containing DHEA.

[Study of the interrelations of ethmozine, cordarone and phenycaberan with heparin].

PubMed

Tolstopiatov, B I

1981-01-01

Cordarone, etmozin and phenycaberan form complexes with heparin. Etmozin and phenycaberan form complexes insoluble in an aqueous medium and exhibit a pronounced antiheparin action in in-vitro experiments. Cordarone and heparin form a complex which is soluble in an aqueous medium. This complex potentiates the biological activity of the anticoagulant. In experiments on rabbits cordarone and phenycaberan increase plasma tolerance to heparin followed by its lowering as compared with controls in experiments with phenycaberan. Etmozin decreases plasma tolerance to heparin.

Process for the enhanced capture of heavy metal emissions

DOEpatents

Biswas, Pratim; Wu, Chang-Yu

2001-01-01

This invention is directed to a process for forming a sorbent-metal complex. The process includes oxidizing a sorbent precursor and contacting the sorbent precursor with a metallic species. The process further includes chemically reacting the sorbent precursor and the metallic species, thereby forming a sorbent-metal complex. In one particular aspect of the invention, at least a portion of the sorbent precursor is transformed into sorbent particles during the oxidation step. These sorbent particles then are contacted with the metallic species and chemically reacted with the metallic species, thereby forming a sorbent-metal complex. Another aspect of the invention is directed to a process for forming a sorbent metal complex in a combustion system. The process includes introducing a sorbent precursor into a combustion system and subjecting the sorbent precursor to an elevated temperature sufficient to oxidize the sorbent precursor and transform the sorbent precursor into sorbent particles. The process further includes contacting the sorbent particles with a metallic species and exposing the sorbent particles and the metallic species to a complex-forming temperature whereby the metallic species reacts with the sorbent particles thereby forming a sorbent-metal complex under UV irradiation.

Phosphate barrier on pore-filled cation-exchange membrane for blocking complexing ions in presence of non-complexing ions

NASA Astrophysics Data System (ADS)

Chavan, Vivek; Agarwal, Chhavi; Shinde, Rakesh N.

2018-06-01

In present work, an approach has been used to form a phosphate groups bearing surface barrier on a cation-exchange membrane (CEM). Using optimized conditions, the phosphate bearing monomer bis[2-(methacryloyloxy)ethyl] phosphate has been grafted on the surface of the host poly(ethersulfone) membranes using UV light induced polymerization. The detailed characterizations have shown that less than a micron layer of phosphate barrier is formed without disturbing the original microporous structure of the host membrane. The pores of thus formed membrane have been blocked by cationic-gel formed by in situ UV-initiator induced polymerization of 2-acrylamido-2-methyl-1-propane sulphonic acid along with crosslinker ethylene glycol dimethacrylate in the pores of the membrane. UV-initiator is required for pore-filling as UV light would not penetrate the interior matrix of the membrane. The phosphate functionalized barrier membrane has been examined for permselectivity using a mixture of representative complexing Am3+ ions and non-complexing Cs+ ions. This experiment has demonstrated that complex forming Am3+ ions are blocked by phosphate barrier layer while non-complexing Cs+ ions are allowed to pass through the channels formed by the crosslinked cationic gel.

Ink composition for making a conductive silver structure

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

Walker, Steven B.; Lewis, Jennifer A.

An ink composition for making a conductive silver structure comprises a silver salt and a complex of (a) a complexing agent and a short chain carboxylic acid or (b) a complexing agent and a salt of a short chain carboxylic acid, according to one embodiment. A method for making a silver structure entails combining a silver salt and a complexing agent, and then adding a short chain carboxylic acid or a salt of the short chain carboxylic acid to the combined silver salt and a complexing agent to form an ink composition. A concentration of the complexing agent in themore » ink composition is reduced to form a concentrated formulation, and the silver salt is reduced to form a conductive silver structure, where the concentrated formulation and the conductive silver structure are formed at a temperature of about 120.degree. C. or less.« less

Erythrocyte haemolysate interacts with ATP-Fe to form a complex containing iron, ATP and 13 800 MW polypeptide.

PubMed

Weaver, J; Zhan, H; Pollack, S

1993-01-01

Iron first entering the reticulocyte is bound to ATP in the low MW cytosolic pool; some is also 'loosely bound' to haemoglobin, coeluting with haemoglobin from a molecular sieve column though not incorporated into haem. When haemolysate is mixed with ATP-Fe in vitro a similar high MW iron-containing complex is formed: the ATP-Fe interacts with a non-haemoglobin constituent of the haemolysate to form a high MW ATP-Fe complex in which the ratio of ATP:Fe (originally 6:1) is reversed, so that the complex contains more iron than ATP. The high MW ATP-Fe complex is formed even when ATP is in 150-fold molar excess and is formed without detectable hydrolysis of the ATP. The activity of haemolysate in forming the high MW ATP-Fe complex is not diminished by dialysis; all of the activity is recovered in the haemoglobin-containing fraction obtained from an Ultrogel AcA 44 column. The activity does not derive from haemoglobin since 85% of the activity is removed when haemoglobin is purified from haemolysate with DEAE-Sephadex. The chelatable iron pool of the cell probably includes both the high MW ATP-Fe complex and low MW ATP-Fe. Shunting of ATP-Fe to a high MW aggregate reduces the amount of iron present in the highly reactive low MW form and thus probably serves to limit the formation of cell damaging radicals.

Reclamation with Recovery of Radionuclides and Toxic Metals from Contaminated Materials, Soils, and Wastes

NASA Technical Reports Server (NTRS)

Francis, A. J.; Dodge, C. J.

1993-01-01

A process has been developed at Brookhaven National Laboratory (BNL) for the removal of metals and radionuclides from contaminated materials, soils, and waste sites. In this process, citric acid, a naturally occurring organic complexing agent, is used to extract metals such as Ba, Cd, Cr, Ni, Zn, and radionuclides Co, Sr, Th, and U from solid wastes by formation of water soluble, metal-citrate complexes. Citric acid forms different types of complexes with the transition metals and actinides, and may involve formation of a bidentate, tridentate, binuclear, or polynuclear complex species. The extract containing radionuclide/metal complex is then subjected to microbiological degradation followed by photochemical degradation under aerobic conditions. Several metal citrate complexes are biodegraded, and the metals are recovered in a concentrated form with the bacterial biomass. Uranium forms binuclear complex with citric acid and is not biodegraded. The supernatant containing uranium citrate complex is separated and upon exposure to light, undergoes rapid degradation resulting in the formation of an insoluble, stable polymeric form of uranium. Uranium is recovered as a precipitate (polyuranate) in a concentrated form for recycling or for appropriate disposal. This treatment process, unlike others which use caustic reagents, does not create additional hazardous wastes for disposal and causes little damage to soil which can then be returned to normal use.

Structure of the active form of human origin recognition complex and its ATPase motor module

PubMed Central

Tocilj, Ante; On, Kin Fan; Yuan, Zuanning; Sun, Jingchuan; Elkayam, Elad; Li, Huilin; Stillman, Bruce; Joshua-Tor, Leemor

2017-01-01

Binding of the Origin Recognition Complex (ORC) to origins of replication marks the first step in the initiation of replication of the genome in all eukaryotic cells. Here, we report the structure of the active form of human ORC determined by X-ray crystallography and cryo-electron microscopy. The complex is composed of an ORC1/4/5 motor module lobe in an organization reminiscent of the DNA polymerase clamp loader complexes. A second lobe contains the ORC2/3 subunits. The complex is organized as a double-layered shallow corkscrew, with the AAA+ and AAA+-like domains forming one layer, and the winged-helix domains (WHDs) forming a top layer. CDC6 fits easily between ORC1 and ORC2, completing the ring and the DNA-binding channel, forming an additional ATP hydrolysis site. Analysis of the ATPase activity of the complex provides a basis for understanding ORC activity as well as molecular defects observed in Meier-Gorlin Syndrome mutations. DOI: http://dx.doi.org/10.7554/eLife.20818.001 PMID:28112645

Structural, luminescence, thermodynamic and theoretical studies on mononuclear complexes of Eu(III) with pyridine monocarboxylate-N-oxides in aqueous solution

NASA Astrophysics Data System (ADS)

Dumpala, Rama Mohana Rao; Rawat, Neetika; Boda, Anil; Ali, Sk. Musharaf; Tomar, B. S.

2018-02-01

The mononuclear complexes formed by Eu(III) with three isomeric pyridine monocarboxylate-N-oxides namely picolinic acid-N-oxide (PANO), nicotinic acid-N-oxide (NANO) and isonicotinic acid-N-oxide (IANO) in aqueous solutions were studied by potentiometry, luminescence spectroscopy and isothermal titration calorimetry (ITC) to determine the speciation, coordination, luminescence properties and thermodynamic parameters of the complexes formed during the course of the reaction. More stable six membered chelate complexes with stoichiometry (MLi, i = 1-4) are formed by Eu(III) with PANO while non chelating ML and ML2 complexes are formed by NANO and IANO. The stability of Eu(III) complexes follow the order PANO > IANO > NANO. The ITC studies inferred an endothermic and innersphere complex formation of Eu(III)-PANO and Eu(III)-IANO whereas an exothermic and outer-sphere complex formation for Eu(III)-NANO. The luminescence life time data further supported the ITC results. Density functional theoretical calculations were carried out to optimize geometries of the complexes and to estimate the energies, structural parameters (bond distances, bond angles) and charges on individual atoms of the same. Theoretical approximations are found to be in good agreement with the experimental observations.

Evolution of complex fruiting-body morphologies in homobasidiomycetes.

PubMed Central

Hibbett, David S; Binder, Manfred

2002-01-01

The fruiting bodies of homobasidiomycetes include some of the most complex forms that have evolved in the fungi, such as gilled mushrooms, bracket fungi and puffballs ('pileate-erect') forms. Homobasidiomycetes also include relatively simple crust-like 'resupinate' forms, however, which account for ca. 13-15% of the described species in the group. Resupinate homobasidiomycetes have been interpreted either as a paraphyletic grade of plesiomorphic forms or a polyphyletic assemblage of reduced forms. The former view suggests that morphological evolution in homobasidiomycetes has been marked by independent elaboration in many clades, whereas the latter view suggests that parallel simplification has been a common mode of evolution. To infer patterns of morphological evolution in homobasidiomycetes, we constructed phylogenetic trees from a dataset of 481 species and performed ancestral state reconstruction (ASR) using parsimony and maximum likelihood (ML) methods. ASR with both parsimony and ML implies that the ancestor of the homobasidiomycetes was resupinate, and that there have been multiple gains and losses of complex forms in the homobasidiomycetes. We also used ML to address whether there is an asymmetry in the rate of transformations between simple and complex forms. Models of morphological evolution inferred with ML indicate that the rate of transformations from simple to complex forms is about three to six times greater than the rate of transformations in the reverse direction. A null model of morphological evolution, in which there is no asymmetry in transformation rates, was rejected. These results suggest that there is a 'driven' trend towards the evolution of complex forms in homobasidiomycetes. PMID:12396494

CoMoDo: identifying dynamic protein domains based on covariances of motion.

PubMed

Wieninger, Silke A; Ullmann, G Matthias

2015-06-09

Most large proteins are built of several domains, compact units which enable functional protein motions. Different domain assignment approaches exist, which mostly rely on concepts of stability, folding, and evolution. We describe the automatic assignment method CoMoDo, which identifies domains based on protein dynamics. Covariances of atomic fluctuations, here calculated by an Elastic Network Model, are used to group residues into domains of different hierarchical levels. The so-called dynamic domains facilitate the study of functional protein motions involved in biological processes like ligand binding and signal transduction. By applying CoMoDo to a large number of proteins, we demonstrate that dynamic domains exhibit features absent in the commonly assigned structural domains, which can deliver insight into the interactions between domains and between subunits of multimeric proteins. CoMoDo is distributed as free open source software at www.bisb.uni-bayreuth.de/CoMoDo.html .

Radiolabeling of multimeric neurotensin(8-13) analogs with the short-lived positron emitter fluorine-18.

PubMed

Hultsch, Christina; Berndt, Mathias; Bergmann, Ralf; Wuest, Frank

2007-07-01

Three methods for (18)F-labeling of dimeric and tetrameric neurotensin(8-13) derivatives were evaluated with respect to the labeling yield and the required peptide amounts. Labeling using N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB) gave low radiochemical yield for the dimeric peptides. Coupling of the tetramer with [(18)F]SFB was not successful. High yields were obtained for labeling of the aminooxy-functionalized neurotensin(8-13) dimer using 4-[(18)F]fluorobenzaldehyde ([(18)F]FBA) whilst coupling of the corresponding tetramer gave only low yields. Labeling of sulfydryl-functionalized neurotensin(8-13) derivatives using the maleinimide 4-[(18)F]fluorobenzaldehyde-O-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexyl]-oxime ([(18)F]FBAM) resulted in high radiochemical yields for both, the dimer and the tetramer. Therefore, [(18)F]FBAM seems to be the most suitable (18)F-labeling agent for multivalent neurotensin(8-13) derivatives.

Cooperativity in Monomeric Enzymes with Single Ligand-Binding Sites

PubMed Central

Porter, Carol M.

2011-01-01

Cooperativity is widespread in biology. It empowers a variety of regulatory mechanisms and impacts both the kinetic and thermodynamic properties of macromolecular systems. Traditionally, cooperativity is viewed as requiring the participation of multiple, spatially distinct binding sites that communicate via ligand-induced structural rearrangements; however, cooperativity requires neither multiple ligand binding events nor multimeric assemblies. An underappreciated manifestation of cooperativity has been observed in the non-Michaelis-Menten kinetic response of certain monomeric enzymes that possess only a single ligand-binding site. In this review, we present an overview of kinetic cooperativity in monomeric enzymes. We discuss the primary mechanisms postulated to give rise to monomeric cooperativity and highlight modern experimental methods that could offer new insights into the nature of this phenomenon. We conclude with an updated list of single subunit enzymes that are suspected of displaying cooperativity, and a discussion of the biological significance of this unique kinetic response. PMID:22137502

A non-chromatographic protein purification strategy using Src 3 homology domains as generalized capture domains.

PubMed

Kim, Heejae; Chen, Wilfred

2016-09-20

Protein purification using inverse phase transition of elastin-like polypeptide (ELP) domains is a useful alternative to chromatography. Genetic fusions of ELP domains to various proteins have the ability to reversibly transition between soluble monomers and micron-sized aggregates and this has been used to selectively purify many ELP fusions. Affinity domains can enhance this technology by using specific protein binding domains to enable ELP mediated affinity capture (EMAC) of proteins of interest (POI) that have been fused to corresponding affinity ligands. In this paper, we highlight the use of Src homology 3 (SH3) domains and corresponding peptide ligands in EMAC that have differential binding affinities towards SH3 for efficient capture and elution of proteins. Furthermore, differences between capture and elution of a monomeric and a multimeric protein were also studied. Copyright © 2016 Elsevier B.V. All rights reserved.

Immunological Properties of Hepatitis B Core Antigen Fusion Proteins

NASA Astrophysics Data System (ADS)

Francis, Michael J.; Hastings, Gillian Z.; Brown, Alan L.; Grace, Ken G.; Rowlands, David J.; Brown, Fred; Clarke, Berwyn E.

1990-04-01

The immunogenicity of a 19 amino acid peptide from foot-and-mouth disease virus has previously been shown to approach that of the inactivated virus from which it was derived after multimeric particulate presentation as an N-terminal fusion with hepatitis B core antigen. In this report we demonstrate that rhinovirus peptide-hepatitis B core antigen fusion proteins are 10-fold more immunogenic than peptide coupled to keyhole limpet hemocyanin and 100-fold more immunogenic than uncoupled peptide with an added helper T-cell epitope. The fusion proteins can be readily administered without adjuvant or with adjuvants acceptable for human and veterinary application and can elicit a response after nasal or oral dosing. The fusion proteins can also act as T-cell-independent antigens. These properties provide further support for their suitability as presentation systems for "foreign" epitopes in the development of vaccines.

Computational and experimental analysis of DNA shuffling

PubMed Central

Maheshri, Narendra; Schaffer, David V.

2003-01-01

We describe a computational model of DNA shuffling based on the thermodynamics and kinetics of this process. The model independently tracks a representative ensemble of DNA molecules and records their states at every stage of a shuffling reaction. These data can subsequently be analyzed to yield information on any relevant metric, including reassembly efficiency, crossover number, type and distribution, and DNA sequence length distributions. The predictive ability of the model was validated by comparison to three independent sets of experimental data, and analysis of the simulation results led to several unique insights into the DNA shuffling process. We examine a tradeoff between crossover frequency and reassembly efficiency and illustrate the effects of experimental parameters on this relationship. Furthermore, we discuss conditions that promote the formation of useless “junk” DNA sequences or multimeric sequences containing multiple copies of the reassembled product. This model will therefore aid in the design of optimal shuffling reaction conditions. PMID:12626764

Process for functionalizing alkanes

DOEpatents

Bergman, R.G.; Janowicz, A.H.; Periana-Pillai, R.A.

1984-06-12

Process for functionalizing saturated hydrocarbons selectively in the terminal position comprises: (a) reacting said saturated hydrocarbons with a metal complex CpRhPMe/sub 3/H/sub 2/ in the presence of ultraviolet radiation at -60/sup 0/ to -17/sup 0/C to form a hydridoalkyl complex CpRhPMe/sub 3/RH; (b) reacting said hydridoalkyl complex with a haloform CHX/sub 3/ at -60/sup 0/ to -17/sup 0/C to form the corresponding haloalkyl complex of step (a) CpRhPMe/sub 3/RX; and (c) reacting said haloalkyl complex with halogen -60 to 25/sup 0/C to form a functional haloalkyl compound.

Reconstitution of a physical complex between the N-formyl chemotactic peptide receptor and G protein. Inhibition by pertussis toxin-catalyzed ADP ribosylation.

PubMed

Bommakanti, R K; Bokoch, G M; Tolley, J O; Schreiber, R E; Siemsen, D W; Klotz, K N; Jesaitis, A J

1992-04-15

Photoaffinity-labeled N-formyl chemotactic peptide receptors from human neutrophils solubilized in octyl glucoside exhibit two forms upon sucrose density gradient sedimentation, with apparent sedimentation coefficients of approximately 4 and 7 S. The 7 S form can be converted to the 4 S form by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with an EC50 of approximately 20 nM, suggesting that the 7 S form may represent a physical complex of the receptor with endogenous G protein (Jesaitis, A. J., Tolley, J. O., Bokoch, G. M., and Allen, R. A. (1989) J. Cell Biol. 109, 2783-2790). To probe the nature of the 7 S form, we reconstituted the 7 S form from the 4 S form by adding purified G protein. The 4 S form, obtained by solubilizing GTP gamma S-treated neutrophil plasma membranes, was incubated with purified (greater than 95%) Gi protein from bovine brain (containing both Gi alpha 1 and Gi alpha 2) or with neutrophil G protein (Gn), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC50 of 7 S complex formation induced by the two G proteins was 70 +/- 25 and 170 +/- 40 nM for Gn and Gi, respectively. No complexation was measurable when bovine transducin (Gt) was used up to 30 times the EC50 for Gn. The EC50 for Gi was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 microM GTP gamma S to the reconstituted 7 S complex caused a complete revision of the receptor to the 4 S form, and anti-Gi peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gi prevented formation of the 7 S form even at 20 times the concentration of unribosylated Gi normally used to attain 50% conversion to the 7 S form. These observations suggest that the 7 S species is a physical complex containing N-formyl chemotactic peptide receptor and G protein.

Properties of lotus seed starch-glycerin monostearin complexes formed by high pressure homogenization.

PubMed

Chen, Bingyan; Zeng, Shaoxiao; Zeng, Hongliang; Guo, Zebin; Zhang, Yi; Zheng, Baodong

2017-07-01

Starch-lipid complexes were prepared using lotus seed starch (LS) and glycerin monostearate (GMS) via a high pressure homogenization (HPH) process, and the effect of HPH on the physicochemical properties of LS-GMS complexes was investigated. The results of Fourier transform infrared spectroscopy and complex index analysis showed that LS-GMS complexes were formed at 40MPa by HPH and the complex index increased with the increase of homogenization pressure. Scanning electron microscopy displayed LS-GMS complexes present more nest-shape structure with increasing homogenization pressure. X-ray diffraction and differential scanning calorimetry results revealed that V-type crystalline polymorph was formed between LS and GMS, with higher homogenization pressure producing an increasingly stable complex. LS-GMS complex inhibited starch granules swelling, solubility and pasting development, which further reduced peak and breakdown viscosity. During storage, LS-GMS complexes prepared by 70-100MPa had higher Avrami exponent values and lower recrystallization rates compared with native starch, which suggested a lower retrogradation trendency. Copyright © 2017 Elsevier Ltd. All rights reserved.

Polyelectrolyte complexes between (cross-linked) N-carboxyethylchitosan and (quaternized) poly[2-(dimethylamino)ethyl methacrylate]: preparation, characterization, and antibacterial properties.

PubMed

Yancheva, Elena; Paneva, Dilyana; Maximova, Vera; Mespouille, Laetitia; Dubois, Philippe; Manolova, Nevena; Rashkov, Iliya

2007-03-01

Novel polyelectrolyte complexes (PECs) between N-carboxyethylchitosan (CECh) and well-defined (quaternized) poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) have been obtained. The modification of chitosan into CECh allows the preparation of PECs in a pH range in which chitosan cannot form complexes. The CECh/PDMAEMA complex is formed in a narrow pH range around 7. The quaternization of the tertiary amino groups of PDMAEMA enables complex formation with CECh both in neutral and in alkaline medium. Cross-linked CECh is also capable of forming complexes with (quaternized) PDMAEMA. The antibacterial activity of (cross-linked) CECh, (quaternized) PDMAEMA, and their complexes against Escherichia coli has been evaluated. In contrast to (quaternized) PDMAEMA, (cross-linked) CECh exhibits no antibacterial activity. The complex formation between cross-linked CECh and (quaternized) PDMAEMA results in a loss of the inherent antibacterial activity of the latter in neutral medium. In acidic medium, the complexes exhibit strong antibacterial activity due to complex disintegration and release of (quaternized) PDMAEMA.

Physical characteristics of the gonadotropin receptor-hormone complexes formed in vivo and in vitro.

PubMed Central

Dufau, M L; Podesta, E J; Catt, K J

1975-01-01

The physical properties of detergent-solubilized gonadotropin receptor-hormone complexes, determined by density gradient centrifugation and gel filtration, were compared after in vivo and in vitro labeling of specific ovarian binding sites with radioiodinated human chorionic gonadotropin (hCG). Following intravenous administration of biologically active 125I-labeled hCG, up to 50% of the gonadotropin tracer was bound to the luteinized ovaries of immature female rats treated with pregnant mare serum/human chorionic gonadotropin. Comparable binding of 125I-labeled hCG was observed after equilibration of ovarian particles with the labeled hormone in vitro. The sedimentation properties of the solubilized receptor-hormone complexes formed in vivo were identical with those derived for the corresponding complexes formed in vitro and extracted with Triton X-100 and Lubrol PX, with sedimentation constants of 8.8 S for the Triton-solubilized complex and 7.0 S for the complex extracted with Lubrol PX. During analytical gel filtration of the Triton-solubilized receptor-hormone complex on Sepharose 6B in 0.1% Triton X-100, the partition coefficient (Kav) of the "in vivo" complex (0.32) was not significantly different from that of the complex formed in vitro (0.29). Gel filtration of the Lubrol-solubilized ovarian particles on Sepharose 6B in 0.5% Lubrol PX gave Kav values for the "in vivo" and "in vitro" labeled complexes of 0.36 and 0.32, respectively. These findings demonstrate that the physical properties of size and shape which determine the partition coefficient and sedimentation characteristics of detergent-solubilized gonadotropin receptor-hormone complexes formed in vitro are not distinguishable from those of the complexes extracted after specific interaction of the ovarian gonadotropin receptors with radioiodinated hCG in vivo. PMID:165502

Synthesis, characterization, DNA-binding and cleavage studies of polypyridyl copper(II) complexes

NASA Astrophysics Data System (ADS)

Gubendran, Ammavasi; Rajesh, Jegathalaprathaban; Anitha, Kandasamy; Athappan, Periyakaruppan

2014-10-01

Six new mixed-ligand copper(II) complexes were synthesized namely [Cu(phen)2OAc]ClO4ṡH2O(1), [Cu(bpy)2OAc]ClO4ṡH2O(2), [Cu(o-ampacac)(phen)]ClO4(3), [Cu(o-ampbzac)(phen)]ClO4(4), [Cu(o-ampacac)(bpy)]ClO4(5), and [Cu(o-ampbzac)(bpy)]ClO4(6) (phen = 1,10-phenanthroline, bpy = 2, 2‧-bipyridine, o-ampacac = (Z)-4-(2-hydroxylamino)pent-3-ene-2-one,o-ampbzac = (Z)-4-(2-hydroxylamino)-4-phenylbut-3-ene-2-one)and characterized by UV-Vis, IR, EPR and cyclic voltammetry. Ligands were characterized by NMR spectra. Single crystal X-ray studies of the complex 1 shows Cu(II) ions are located in a highly distorted octahedral environment. Absorption spectral studies reveal that the complexes 1-6 exhibit hypochromicity during the interaction with DNA and binding constant values derived from spectral and electrochemical studies indicate that complexes 1, 2 and 3 bind strongly with DNA possibly by an intercalative mode. Electrochemical studies reveal that the complexes 1-4 prefer to bind with DNA in Cu(I) rather than Cu(II) form. The shift in the formal potentials E1/2 and CD spectral studies suggest groove or electrostatic binding mode for the complexes 4-6. Complex 1 can cleave supercoiled (SC) pUC18 DNA efficiently into nicked form II under photolytic conditions and into an open circular form (form II) and linear form (form III) in the presence of H2O2 at pH 8.0 and 37 °C, while the complex 2 does not cleave DNA under similar conditions.

Characterization of the interaction between subunits of the botulinum toxin complex produced by serotype D through tryptic susceptibility of the isolated components and complex forms.

PubMed

Suzuki, Tomonori; Watanabe, Toshihiro; Mutoh, Shingo; Hasegawa, Kimiko; Kouguchi, Hirokazu; Sagane, Yoshimasa; Fujinaga, Yukako; Oguma, Keiji; Ohyama, Tohru

2005-05-01

The 650 kDa large toxin complex (L-TC) produced by Clostridium botulinum serotype D strain 4947 (D-4947) has a subunit structure composed of unnicked components, i.e. neurotoxin (NT), non-toxic non-haemagglutinin (NTNHA) and three haemagglutinin subcomponents (HA-70, HA-33 and HA-17). In this study, subunit interactions were investigated through the susceptibilities of the toxin components to limited trypsin proteolysis. Additionally, complex forms were reconstituted in vitro by various combinations of individual components. Trypsin treatment of intact D-4947 L-TC led to the formation of mature L-TC with nicks at specific sites of each component, which is usually observed in other strains of serotype D. NT, NTNHA and HA-17 were cleaved at their specific sites in either the single or complex forms, but HA-33 showed no sign of proteolysis. Unlike the other components, HA-70 was digested into random fragments as a single form, but it was cleaved into two fragments in the complex form. Based on the relative position of exposed or hidden regions of the individual components in the complex derived from their tryptic susceptibilities, an assembly model is proposed for the arrangement of individual subunits in the botulinum L-TC.

The Roles of APOBEC3G Complexes in the Incorporation of APOBEC3G into HIV-1

PubMed Central

Zhang, Quan; Liu, Zhenlong; Jia, Pingping; Zhou, Jinming; Guo, Fei; You, Xuefu; Yu, Liyan; Zhao, Lixun; Jiang, Jiandong; Cen, Shan

2013-01-01

Background The incorporation of human APOBEC3G (hA3G) into HIV is required for exerting its antiviral activity, therefore the mechanism underlying hA3G virion encapsidation has been investigated extensively. hA3G was shown to form low-molecular-mass (LMM) and high-molecular-mass (HMM) complexes. The function of different forms of hA3G in its viral incorporation remains unclear. Methodology/Principal Findings In this study, we investigated the subcellular distribution and lipid raft association of hA3G using subcellular fractionation, membrane floatation assay and pulse-chase radiolabeling experiments respectively, and studied the correlation between the ability of hA3G to form the different complex and its viral incorporation. Our work herein provides evidence that the majority of newly-synthesized hA3G interacts with membrane lipid raft domains to form Lipid raft-associated hA3G (RA hA3G), which serve as the precursor of mature HMM hA3G complex, while a minority of newly-synthesized hA3G remains in the cytoplasm as a soluble LMM form. The distribution of hA3G among the soluble LMM form, the RA LMM form and the mature forms of HMM is regulated by a mechanism involving the N-terminal part of the linker region and the C-terminus of hA3G. Mutagenesis studies reveal a direct correlation between the ability of hA3G to form the RA LMM complex and its viral incorporation. Conclusions/Significance Together these data suggest that the Lipid raft-associated LMM A3G complex functions as the cellular source of viral hA3G. PMID:24098356