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Sample records for protein subunit interfaces

  1. The subunit interfaces of weakly associated homodimeric proteins.

    PubMed

    Dey, Sucharita; Pal, Arumay; Chakrabarti, Pinak; Janin, Joël

    2010-04-23

    We analyzed subunit interfaces in 315 homodimers with an X-ray structure in the Protein Data Bank, validated by checking the literature for data that indicate that the proteins are dimeric in solution and that, in the case of the "weak" dimers, the homodimer is in equilibrium with the monomer. The interfaces of the 42 weak dimers, which are smaller by a factor of 2.4 on average than in the remainder of the set, are comparable in size with antibody-antigen or protease-inhibitor interfaces. Nevertheless, they are more hydrophobic than in the average transient protein-protein complex and similar in amino acid composition to the other homodimer interfaces. The mean numbers of interface hydrogen bonds and hydration water molecules per unit area are also similar in homodimers and transient complexes. Parameters related to the atomic packing suggest that many of the weak dimer interfaces are loosely packed, and we suggest that this contributes to their low stability. To evaluate the evolutionary selection pressure on interface residues, we calculated the Shannon entropy of homologous amino acid sequences at 60% sequence identity. In 93% of the homodimers, the interface residues are better conserved than the residues on the protein surface. The weak dimers display the same high degree of interface conservation as other homodimers, but their homologs may be heterodimers as well as homodimers. Their interfaces may be good models in terms of their size, composition, and evolutionary conservation for the labile subunit contacts that allow protein assemblies to share and exchange components, allosteric proteins to undergo quaternary structure transitions, and molecular machines to operate in the cell.

  2. "Silent" Amino Acid Residues at Key Subunit Interfaces Regulate the Geometry of Protein Nanocages.

    PubMed

    Zhang, Shengli; Zang, Jiachen; Zhang, Xiaorong; Chen, Hai; Mikami, Bunzo; Zhao, Guanghua

    2016-11-22

    Rendering the geometry of protein-based assemblies controllable remains challenging. Protein shell-like nanocages represent particularly interesting targets for designed assembly. Here, we introduce an engineering strategy-key subunit interface redesign (KSIR)-that alters a natural subunit-subunit interface by selective deletion of a small number of "silent" amino acid residues (no participation in interfacial interactions) into one that triggers the generation of a non-native protein cage. We have applied KSIR to construct a non-native 48-mer nanocage from its native 24-mer recombinant human H-chain ferritin (rHuHF). This protein is a heteropolymer composed of equal numbers of two different subunits which are derived from one polypeptide. This strategy has allowed the study of conversion between protein nanocages with different geometries by re-engineering key subunit interfaces and the demonstration of the important role of the above-mentioned specific residues in providing geometric specificity for protein assembly.

  3. NMR Insight into Myosin-Binding Subunit Coiled Coil Structure Reveals Binding Interface with Protein Kinase G-Iα Leucine Zipper in Vascular Function.

    PubMed

    Sharma, Alok K; Birrane, Gabriel G; Anklin, Clemens; Rigby, Alan C; Alper, Seth L

    2017-03-09

    Nitrovasodilators relax vascular smooth muscle cells (VSMC) in part by modulating the interaction of the C-terminal coiled-coil domain (CC) and/or leucine zipper (LZ) domain of the myosin light-chain phosphatase (MLCP) component, myosin-binding subunit (MBS), with the N-terminal LZ domain of protein kinase G (PKG)-Iα. Despite the importance of vasodilation in cardiovascular homeostasis and therapy, our structural understanding of the MBS CC interaction with LZ PKG-Iα has remained limited. Here, we report the three-dimensional NMR solution structure of homodimeric CC MBS in which aa 932-967 form a coiled-coil of two monomeric α-helices in parallel orientation. We found that the structure is stabilized by non-covalent interactions, with dominant contributions from hydrophobic residues at a and d heptad positions. Using NMR chemical shift perturbation (CSP) analysis, we identified a subset of hydrophobic and charged residues of CC MBS (localized within and adjacent to the C-terminal region) contributing to the dimer-dimer interaction interface between homodimeric CC MBS and homodimeric LZ PKG-Iα. 15N backbone relaxation NMR revealed the dynamic features of the CC MBS interface residues identified by NMR CSP. Paramagnetic relaxation-enhancement (PRE) and CSP NMR guided HADDOCK modeling of the dimer-dimer interface of the hetero-tetrameric complex exhibits the involvement of non-covalent intermolecular interactions that are localized within and adjacent to the C-terminal regions of each homodimer. These results deepen our understanding of the binding restraints of this CC MBS-LZ PKG-Iα low-affinity heterotetrameric complex and allow re-evaluation of the role(s) of MLCP partner polypeptides in regulation of VSMC contractility.

  4. The multifaceted subunit interfaces of ionotropic glutamate receptors.

    PubMed

    Green, Tim; Nayeem, Naushaba

    2015-01-01

    The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research.

  5. The multifaceted subunit interfaces of ionotropic glutamate receptors.

    PubMed

    Green, Tim; Nayeem, Naushaba

    2014-06-06

    The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research.

  6. Protein synthesis by ribosomes with tethered subunits.

    PubMed

    Orelle, Cédric; Carlson, Erik D; Szal, Teresa; Florin, Tanja; Jewett, Michael C; Mankin, Alexander S

    2015-08-06

    The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established model of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed to be essential for biogenesis, since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation. Here we show that ribosomes with tethered and thus inseparable subunits (termed Ribo-T) are capable of successfully carrying out protein synthesis. By engineering a hybrid rRNA composed of both small and large subunit rRNA sequences, we produced a functional ribosome in which the subunits are covalently linked into a single entity by short RNA linkers. Notably, Ribo-T was not only functional in vitro, but was also able to support the growth of Escherichia coli cells even in the absence of wild-type ribosomes. We used Ribo-T to create the first fully orthogonal ribosome-messenger RNA system, and demonstrate its evolvability by selecting otherwise dominantly lethal rRNA mutations in the peptidyl transferase centre that facilitate the translation of a problematic protein sequence. Ribo-T can be used for exploring poorly understood functions of the ribosome, enabling orthogonal genetic systems, and engineering ribosomes with new functions.

  7. Protein-Protein Interfaces in Viral Capsids Are Structurally Unique.

    PubMed

    Cheng, Shanshan; Brooks, Charles L

    2015-11-06

    Viral capsids exhibit elaborate and symmetrical architectures of defined sizes and remarkable mechanical properties not seen with cellular macromolecular complexes. Given the uniqueness of the higher-order organization of viral capsid proteins in the virosphere, we explored the question of whether the patterns of protein-protein interactions within viral capsids are distinct from those in generic protein complexes. Our comparative analysis involving a non-redundant set of 551 inter-subunit interfaces in viral capsids from VIPERdb and 20,014 protein-protein interfaces in non-capsid protein complexes from the Protein Data Bank found 418 generic protein-protein interfaces that share similar physicochemical patterns with some protein-protein interfaces in the capsid set, using the program PCalign we developed for comparing protein-protein interfaces. This overlap in the structural space of protein-protein interfaces is significantly small, with a p-value <0.0001, based on a permutation test on the total set of protein-protein interfaces. Furthermore, the generic protein-protein interfaces that bear similarity in their spatial and chemical arrangement with capsid ones are mostly small in size with fewer than 20 interfacial residues, which results from the relatively limited choices of natural design for small interfaces rather than having significant biological implications in terms of functional relationships. We conclude based on this study that protein-protein interfaces in viral capsids are non-representative of patterns in the smaller, more compact cellular protein complexes. Our finding highlights the design principle of building large biological containers from repeated, self-assembling units and provides insights into specific targets for antiviral drug design for improved efficacy.

  8. Cooperative protein structural dynamics of homodimeric hemoglobin linked to water cluster at subunit interface revealed by time-resolved X-ray solution scattering

    PubMed Central

    Kim, Jong Goo; Muniyappan, Srinivasan; Oang, Key Young; Kim, Tae Wu; Yang, Cheolhee; Kim, Kyung Hwan; Kim, Jeongho; Ihee, Hyotcherl

    2016-01-01

    Homodimeric hemoglobin (HbI) consisting of two subunits is a good model system for investigating the allosteric structural transition as it exhibits cooperativity in ligand binding. In this work, as an effort to extend our previous study on wild-type and F97Y mutant HbI, we investigate structural dynamics of a mutant HbI in solution to examine the role of well-organized interfacial water cluster, which has been known to mediate intersubunit communication in HbI. In the T72V mutant of HbI, the interfacial water cluster in the T state is perturbed due to the lack of Thr72, resulting in two less interfacial water molecules than in wild-type HbI. By performing picosecond time-resolved X-ray solution scattering experiment and kinetic analysis on the T72V mutant, we identify three structurally distinct intermediates (I1, I2, and I3) and show that the kinetics of the T72V mutant are well described by the same kinetic model used for wild-type and F97Y HbI, which involves biphasic kinetics, geminate recombination, and bimolecular CO recombination. The optimized kinetic model shows that the R-T transition and bimolecular CO recombination are faster in the T72V mutant than in the wild type. From structural analysis using species-associated difference scattering curves for the intermediates, we find that the T-like deoxy I3 intermediate in solution has a different structure from deoxy HbI in crystal. In addition, we extract detailed structural parameters of the intermediates such as E-F distance, intersubunit rotation angle, and heme-heme distance. By comparing the structures of protein intermediates in wild-type HbI and the T72V mutant, we reveal how the perturbation in the interfacial water cluster affects the kinetics and structures of reaction intermediates of HbI. PMID:27158635

  9. Subunit interface mutants of rabbit muscle aldolase form active dimers.

    PubMed Central

    Beernink, P. T.; Tolan, D. R.

    1994-01-01

    We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C. Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability. PMID:7833800

  10. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function.

    PubMed

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V; Craig, Patricio O; Millar, Neil S; Bouzat, Cecilia; Elgoyhen, Ana Belén

    2017-03-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits.

  11. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function

    PubMed Central

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K.; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V.; Craig, Patricio O.; Millar, Neil S.; Bouzat, Cecilia

    2017-01-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits. PMID:28069778

  12. Intersurf: dynamic interface between proteins.

    PubMed

    Ray, Nicolas; Cavin, Xavier; Paul, Jean-Claude; Maigret, Bernard

    2005-01-01

    Protein docking is a fundamental biological process that links two proteins. This link is typically defined by an interaction between two large zones of the protein boundaries. Visualizing such an interface is useful to understand the process thanks to 3D protein structures, to estimate the quality of docking simulation results, and to classify interactions in order to predict docking affinity between classes of interacting zones. Since the interface may be defined by a surface that separates the two proteins, it is possible to create a map of interaction that allows comparisons to be performed in 2D. This paper presents a very fast algorithm that extracts an interface surface and creates a valid and low-distorted interaction map. Another benefit of our approach is that a pre-computed part of the algorithm enables the surface to be updated in real-time while residues are moved.

  13. Subunit Interactions at the V1-Vo Interface in Yeast Vacuolar ATPase*

    PubMed Central

    Oot, Rebecca A.; Wilkens, Stephan

    2012-01-01

    Eukaryotic vacuolar ATPase (V-ATPase) is regulated by a reversible dissociation mechanism that involves breaking and reforming of protein-protein interactions at the interface of the V1-ATPase and Vo-proton channel domains. We found previously that the head domain of the single copy C subunit (Chead) binds one subunit EG heterodimer with high affinity (Oot, R.A. and Wilkens, S. (2010) J. Biol. Chem. 285, 24654–24664). Here we generated a water-soluble construct of the N-terminal domain of the Vo “a” subunit composed of amino acid residues 104–372 (aNT(104–372)). Analytical gel filtration chromatography and sedimentation velocity analysis revealed that aNT(104–372) undergoes reversible dimerization in a concentration-dependent manner. A low-resolution molecular envelope was calculated for the aNT(104–372) dimer using small angle x-ray scattering data. Isothermal titration calorimetry experiments revealed that aNT(104–372) binds the Cfoot and EG heterodimer with dissociation constants of 22 and 33 μm, respectively. We speculate that the spatial closeness of the aNT, Cfoot, and EG binding sites in the intact V-ATPase results in a high-avidity interaction that is able to resist the torque of rotational catalysis, and that reversible enzyme dissociation is initiated by breaking either the aNT(104–372)-Cfoot or aNT(104–372)-EG interaction by an as-yet unknown signaling mechanism. PMID:22367203

  14. Protein Expression of Proteasome Subunits in Elderly Patients with Schizophrenia

    PubMed Central

    Scott, Madeline R; Rubio, Maria D; Haroutunian, Vahram; Meador-Woodruff, James H

    2016-01-01

    The ubiquitin proteasome system (UPS) is a major regulator of protein processing, trafficking, and degradation. While protein ubiquitination is utilized for many cellular processes, one major function of this system is to target proteins to the proteasome for degradation. In schizophrenia, studies have found UPS transcript abnormalities in both blood and brain, and we have previously reported decreased protein expression of ubiquitin-associated proteins in brain. To test whether the proteasome is similarly dysregulated, we measured the protein expression of proteasome catalytic subunits as well as essential subunits from proteasome regulatory complexes in 14 pair-matched schizophrenia and comparison subjects in superior temporal cortex. We found decreased expression of Rpt1, Rpt3, and Rpt6, subunits of the 19S regulatory particle essential for ubiquitin-dependent degradation by the proteasome. Additionally, the α subunit of the 11S αβ regulatory particle, which enhances proteasomal degradation of small peptides and unfolded proteins, was also decreased. Haloperidol-treated rats did not have altered expression of these subunits, suggesting the changes we observed in schizophrenia are likely not due to chronic antipsychotic treatment. Interestingly, expression of the catalytic subunits of both the standard and immunoproteasome were unchanged, suggesting the abnormalities we observed may be specific to the complexed state of the proteasome. Aging has significant effects on the proteasome, and several subunits (20S β2, Rpn10, Rpn13, 11Sβ, and 11Sγ) were significantly correlated with subject age. These data provide further evidence of dysfunction of the ubiquitin-proteasome system in schizophrenia, and suggest that altered proteasome activity may be associated with the pathophysiology of this illness. PMID:26202105

  15. Subunit Interfaces Contribute Differently to Activation and Allosteric Modulation of Neuronal Nicotinic Acetylcholine Receptors

    PubMed Central

    Short, Caitlin A.; Cao, Angela T.; Wingfield, Molly A.; Doers, Matthew E.; Jobe, Emily M.; Wang, Nan; Levandoski, Mark M.

    2015-01-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in the nervous system and are implicated in many normal and pathological processes. The structural determinants of allostery in nAChRs are not well understood. One class of nAChR allosteric modulators, including the small molecule morantel (Mor), acts from a site that is structurally homologous to the canonical agonist site but exists in the β(+)/α(–) subunit interface. We hypothesized that all nAChR subunits move with respect to each other during channel activation and allosteric modulation. We therefore studied five pairs of residues predicted to span the interfaces of α3β2 receptors, one at the agonist interface and four at the modulator interface. Substituting cysteines in these positions, we used disulfide trapping to perturb receptor function. The pair α3Y168-β2D190, involving the C loop region of the β2 subunit, mediates modulation and agonist activation, because evoked currents were reduced up to 50% following oxidation (H2O2) treatment. The pair α3S125-β2Q39, below the canonical site, is also involved in channel activation, in accord with previous studies of the muscle-type receptor; however, the pair is differentially sensitive to ACh activation and Mor modulation (currents decreased 60% and 80%, respectively). The pairs α3Q37-β2A127 and α3E173-β2R46, both in the non-canonical interface, showed increased currents following oxidation, suggesting that subunit movements are not symmetrical. Together, our results from disulfide trapping and further mutation analysis indicate that subunit interface movement is important for allosteric modulation of nAChRs, but that the two types of interfaces contribute unequally to receptor activation. PMID:25486620

  16. Geometric similarity between protein-RNA interfaces.

    PubMed

    Zhou, Peng; Zou, Jianwei; Tian, Feifei; Shang, Zhicai

    2009-12-01

    A new method is described to measure the geometric similarity between protein-RNA interfaces quantitatively. The method is based on a procedure that dissects the interface geometry in terms of the spatial relationships between individual amino acid nucleotide pairs. Using this technique, we performed an all-on-all comparison of 586 protein-RNA interfaces deposited in the current Protein Data Bank, as the result, an interface-interface similarity score matrix was obtained. Based upon this matrix, hierarchical clustering was carried out which yielded a complete clustering tree for the 586 protein-RNA interfaces. By investigating the organizing behavior of the clustering tree and the SCOP classification of protein partners in complexes, a geometrically nonredundant, diverse data set (representative data set) consisting of 45 distinct protein-RNA interfaces was extracted for the purpose of studying protein-RNA interactions, RNA regulations, and drug design. We classified protein-RNA interfaces into three types. In type I, the families and interface structural classes of the protein partners, as well as the interface geometries are all similar. In type II, the interface geometries and the interface structural classes are similar, whereas the protein families are different. In type III, only the interface geometries are similar but the protein families and the interface structural classes are distinct. Furthermore, we also show two new RNA recognition themes derived from the representative data set.

  17. Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution.

    PubMed

    He, Yi-Ming; Ma, Bin-Guang

    2016-05-25

    Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions.

  18. Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

    PubMed Central

    He, Yi-Ming; Ma, Bin-Guang

    2016-01-01

    Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions. PMID:27220911

  19. Heterotrimeric G protein subunit Gγ13 is critical to olfaction

    PubMed Central

    Li, Feng; Ponissery-Saidu, Samsudeen; Yee, Karen; Wang, Hong; Chen, Meng-Ling; Iguchi, Naoko; Zhang, Genhua; Jiang, Ping; Reisert, Johannes; Huang, Liquan

    2013-01-01

    The activation of G-protein-coupled olfactory receptors on the olfactory sensory neurons (OSNs) triggers a signaling cascade, which is mediated by a heterotrimeric G protein consisting of α, β and γ subunits. Although its α subunit, Gαolf, has been identified and well characterized, the identities of its β and γ subunits and their function in olfactory signal transduction, however, have not been well established yet. We and others have found the expression of Gγ13 in the olfactory epithelium, particularly in the cilia of the OSNs. In this study, we generated a conditional gene knockout mouse line to specifically nullify Gγ13 expression in the olfactory marker protein-expressing OSNs. Immunohistochemical and Western blot results showed that Gγ13 subunit was indeed eliminated in the mutant mice’s olfactory epithelium. Intriguingly, Gαolf, β1 subunits, Ric-8B and CEP290 proteins were also absent in the epithelium whereas the presence of the effector enzyme adenylyl cyclase III remained largely unaltered. Electro-olfactogram studies showed that the mutant animals had greatly reduced responses to a battery of odorants including three presumable pheromones. Behavioral tests indicated that the mutant mice had a remarkably reduced ability to perform an odor-guided search task although their motivation and agility seemed normal. Our results indicate that Gαolf exclusively forms a functional heterotrimeric G protein with Gβ1 and Gγ13 in OSNs, mediating olfactory signal transduction. The identification of the olfactory G protein’s βγ moiety has provided a novel approach to understanding the feedback regulation of olfactory signal transduction pathways as well as the control of subcellular structures of OSNs. PMID:23637188

  20. Genetically encoding a light switch in an ionotropic glutamate receptor reveals subunit-specific interfaces

    PubMed Central

    Zhu, Shujia; Riou, Morgane; Yao, C. Andrea; Carvalho, Stéphanie; Rodriguez, Pamela C.; Bensaude, Olivier; Paoletti, Pierre; Ye, Shixin

    2014-01-01

    Reprogramming receptors to artificially respond to light has strong potential for molecular studies and interrogation of biological functions. Here, we design a light-controlled ionotropic glutamate receptor by genetically encoding a photoreactive unnatural amino acid (UAA). The photo–cross-linker p-azido-l-phenylalanine (AzF) was encoded in NMDA receptors (NMDARs), a class of glutamate-gated ion channels that play key roles in neuronal development and plasticity. AzF incorporation in the obligatory GluN1 subunit at the GluN1/GluN2B N-terminal domain (NTD) upper lobe dimer interface leads to an irreversible allosteric inhibition of channel activity upon UV illumination. In contrast, when pairing the UAA-containing GluN1 subunit with the GluN2A subunit, light-dependent inactivation is completely absent. By combining electrophysiological and biochemical analyses, we identify subunit-specific structural determinants at the GluN1/GluN2 NTD dimer interfaces that critically dictate UV-controlled inactivation. Our work reveals that the two major NMDAR subtypes differ in their ectodomain-subunit interactions, in particular their electrostatic contacts, resulting in GluN1 NTD coupling more tightly to the GluN2B NTD than to the GluN2A NTD. It also paves the way for engineering light-sensitive ligand-gated ion channels with subtype specificity through the genetic code expansion. PMID:24715733

  1. Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the FO sector of Escherichia coli ATP synthase

    PubMed Central

    Hutcheon, Marcus L.; Duncan, Thomas M.; Ngai, Helen; Cross, Richard L.

    2001-01-01

    Subunit rotation within the F1 catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded FO sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the single-copy a subunit (stator). Here we report evidence for an energy-dependent rotation at this interface. FOF1 was expressed with a pair of substituted cysteines positioned to allow an intersubunit disulfide crosslink between subunit a and a c subunit [aN214C/cM65C; Jiang, W. & Fillingame, R. H. (1998) Proc. Natl. Acad. Sci. USA 95, 6607–6612]. Membranes were treated with N,N′-dicyclohexyl-[14C]carbodiimide to radiolabel the D61 residue on less than 20% of the c subunits. After oxidation to form an a–c crosslink, the c subunit properly aligned to crosslink to subunit a was found to contain very little 14C label relative to other members of the c ring. However, exposure to MgATP before oxidation significantly increased the radiolabel in the a–c crosslink, indicating that a different c subunit was now aligned with subunit a. This increase was not induced by exposure to MgADP/Pi. Furthermore, preincubation with MgADP and azide to inhibit F1 or with high concentrations of N,N′-dicyclohexylcarbodiimide to label most c subunits prevented the ATP effect. These results provide evidence for an energy-dependent rotation of the c ring relative to subunit a. PMID:11438702

  2. In Search of Small Molecule Inhibitors Targeting the Flexible CK2 Subunit Interface.

    PubMed

    Bestgen, Benoît; Belaid-Choucair, Zakia; Lomberget, Thierry; Le Borgne, Marc; Filhol, Odile; Cochet, Claude

    2017-02-03

    Protein kinase CK2 is a tetrameric holoenzyme composed of two catalytic (α and/or α') subunits and two regulatory (β) subunits. Crystallographic data paired with fluorescence imaging techniques have suggested that the formation of the CK2 holoenzyme complex within cells is a dynamic process. Although the monomeric CK2α subunit is endowed with a constitutive catalytic activity, many of the plethora of CK2 substrates are exclusively phosphorylated by the CK2 holoenzyme. This means that the spatial and high affinity interaction between CK2α and CK2β subunits is critically important and that its disruption may provide a powerful and selective way to block the phosphorylation of substrates requiring the presence of CK2β. In search of compounds inhibiting this critical protein-protein interaction, we previously designed an active cyclic peptide (Pc) derived from the CK2β carboxy-terminal domain that can efficiently antagonize the CK2 subunit interaction. To understand the functional significance of this interaction, we generated cell-permeable versions of Pc, exploring its molecular mechanisms of action and the perturbations of the signaling pathways that it induces in intact cells. The identification of small molecules inhibitors of this critical interaction may represent the first-choice approach to manipulate CK2 in an unconventional way.

  3. In Search of Small Molecule Inhibitors Targeting the Flexible CK2 Subunit Interface

    PubMed Central

    Bestgen, Benoît; Belaid-Choucair, Zakia; Lomberget, Thierry; Le Borgne, Marc; Filhol, Odile; Cochet, Claude

    2017-01-01

    Protein kinase CK2 is a tetrameric holoenzyme composed of two catalytic (α and/or α’) subunits and two regulatory (β) subunits. Crystallographic data paired with fluorescence imaging techniques have suggested that the formation of the CK2 holoenzyme complex within cells is a dynamic process. Although the monomeric CK2α subunit is endowed with a constitutive catalytic activity, many of the plethora of CK2 substrates are exclusively phosphorylated by the CK2 holoenzyme. This means that the spatial and high affinity interaction between CK2α and CK2β subunits is critically important and that its disruption may provide a powerful and selective way to block the phosphorylation of substrates requiring the presence of CK2β. In search of compounds inhibiting this critical protein–protein interaction, we previously designed an active cyclic peptide (Pc) derived from the CK2β carboxy-terminal domain that can efficiently antagonize the CK2 subunit interaction. To understand the functional significance of this interaction, we generated cell-permeable versions of Pc, exploring its molecular mechanisms of action and the perturbations of the signaling pathways that it induces in intact cells. The identification of small molecules inhibitors of this critical interaction may represent the first-choice approach to manipulate CK2 in an unconventional way. PMID:28165359

  4. Model study of protein unfolding by interfaces

    NASA Astrophysics Data System (ADS)

    Chakarova, S. D.; Carlsson, A. E.

    2004-02-01

    We study interface-induced protein unfolding on hydrophobic and polar interfaces by means of a two-dimensional lattice model and an exhaustive enumeration ground-state structure search, for a set of model proteins of length 20 residues. We compare the effects of the two types of interfaces, and search for criteria that influence the retention of a protein’s native-state structure upon adsorption. We find that the unfolding proceeds by a large, sudden loss of native contacts. The unfolding at polar interfaces exhibits similar behavior to that at hydrophobic interfaces but with a much weaker interface coupling strength. Further, we find that the resistance of proteins to unfolding in our model is positively correlated with the magnitude of the folding energy in the native-state structure, the thermal stability (or energy gap) for that structure, and the interface energy for native-state adsorption. We find these factors to be of roughly equal importance.

  5. Statistical analysis and prediction of protein-protein interfaces.

    PubMed

    Bordner, Andrew J; Abagyan, Ruben

    2005-08-15

    Predicting protein-protein interfaces from a three-dimensional structure is a key task of computational structural proteomics. In contrast to geometrically distinct small molecule binding sites, protein-protein interface are notoriously difficult to predict. We generated a large nonredundant data set of 1494 true protein-protein interfaces using biological symmetry annotation where necessary. The data set was carefully analyzed and a Support Vector Machine was trained on a combination of a new robust evolutionary conservation signal with the local surface properties to predict protein-protein interfaces. Fivefold cross validation verifies the high sensitivity and selectivity of the model. As much as 97% of the predicted patches had an overlap with the true interface patch while only 22% of the surface residues were included in an average predicted patch. The model allowed the identification of potential new interfaces and the correction of mislabeled oligomeric states.

  6. Cornichon proteins determine the subunit composition of synaptic AMPA receptors.

    PubMed

    Herring, Bruce E; Shi, Yun; Suh, Young Ho; Zheng, Chan-Ying; Blankenship, Sabine M; Roche, Katherine W; Nicoll, Roger A

    2013-03-20

    Cornichon-2 and cornichon-3 (CNIH-2/-3) are AMPA receptor (AMPAR) binding proteins that promote receptor trafficking and markedly slow AMPAR deactivation in heterologous cells, but their role in neurons is unclear. Using CNIH-2 and CNIH-3 conditional knockout mice, we find a profound reduction of AMPAR synaptic transmission in the hippocampus. This deficit is due to the selective loss of surface GluA1-containing AMPARs (GluA1A2 heteromers), leaving a small residual pool of synaptic GluA2A3 heteromers. The kinetics of AMPARs in neurons lacking CNIH-2/-3 are faster than those in WT neurons due to the fast kinetics of GluA2A3 heteromers. The remarkably selective effect of CNIHs on the GluA1 subunit is probably mediated by TARP γ-8, which prevents a functional association of CNIHs with non-GluA1 subunits. These results point to a sophisticated interplay between CNIHs and γ-8 that dictates subunit-specific AMPAR trafficking and the strength and kinetics of synaptic AMPAR-mediated transmission.

  7. Differential Localization of G Protein βγ Subunits

    PubMed Central

    2015-01-01

    G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system. PMID:24568373

  8. Effective polymer adjuvants for sustained delivery of protein subunit vaccines.

    PubMed

    Adams, Justin R; Haughney, Shannon L; Mallapragada, Surya K

    2015-03-01

    We have synthesized thermogelling cationic amphiphilic pentablock copolymers that have the potential to act as injectable vaccine carriers and adjuvants that can simultaneously provide sustained delivery and enhance the immunogenicity of released antigen. While these pentablock copolymers have shown efficacy in DNA delivery in past studies, the ability to deliver both DNA and protein for subunit vaccines using the same polymeric carrier can provide greater flexibility and efficacy. We demonstrate the ability of these pentablock copolymers, and the parent triblock Pluronic copolymers to slowly release structurally intact and antigenically stable protein antigens in vitro, create an antigen depot through long-term injection-site persistence and enhance the in vivo immune response to these antigens. We show release of the model protein antigen ovalbumin in vitro from the thermogelling block copolymers with the primary, secondary and tertiary structures of the released protein unchanged compared to the native protein, and its antigenicity preserved upon release. The block copolymers form a gel at physiological temperatures that serves as an antigenic depot and persists in vivo at the site of injection for over 50days. The pentablock copolymers show a significant fivefold enhancement in the immune response compared to soluble protein alone, even 6weeks after the administration, based on measurement of antibody titers. These results demonstrate the potential of these block copolymers hydrogels to persist for several weeks and sustain the release of antigen with minimal effects on protein stability and antigenicity; and their ability to be used simultaneously as a sustained delivery device as well as a subunit vaccine adjuvant platform.

  9. Design of a hyperstable 60-subunit protein icosahedron

    NASA Astrophysics Data System (ADS)

    Hsia, Yang; Bale, Jacob B.; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K.; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N.; Gonen, Tamir; King, Neil P.; Baker, David

    2016-07-01

    The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

  10. Design of a hyperstable 60-subunit protein dodecahedron. [corrected].

    PubMed

    Hsia, Yang; Bale, Jacob B; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N; Gonen, Tamir; King, Neil P; Baker, David

    2016-07-07

    The dodecahedron [corrected] is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The dodecahedron [corrected] is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

  11. Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins*

    PubMed Central

    Yao, Xin-Qiu; Malik, Rabia U.; Griggs, Nicholas W.; Skjærven, Lars; Traynor, John R.; Sivaramakrishnan, Sivaraj; Grant, Barry J.

    2016-01-01

    G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gαi. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function. PMID:26703464

  12. T cells specific for alpha-beta interface regions of hemoglobin recognize the isolated subunit but not the tetramer and indicate presentation without processing.

    PubMed Central

    Atassi, M Z; Yoshioka, M; Bixler, G S

    1989-01-01

    Processing of a protein antigen into fragments is believed to be a prerequisite for its presentation by the antigen-presenting cell to the T cell. This model would predict that, in oligomeric proteins, T cells prepared with specificity for regions that are buried within subunit association surfaces should recognize the respective regions in vitro equally well on the isolated subunit or on the oligomer. Three hemoglobin (Hb) alpha-chain synthetic peptides, corresponding to areas that are situated either completely [alpha-(31-45)] or partially [alpha-(41-45) and alpha-(81-95)] within the interface between the alpha and beta subunits of Hb, and a fourth peptide representing a completely exposed area in tetrameric Hb were used as immunogens in SJL/J (H-2s) mice. Peptide-primed T cells were passaged in vitro with the respective peptide to obtain peptide-specific T-lymphocyte lines. T-cell clones were isolated from these lines by limiting dilution. T-cell lines and clones that were specific for buried regions in the subunit association surfaces recognized the free peptide and the isolated subunit but not the Hb tetramer. On the other hand, T cells with specificity against regions that are not involved in subunit interaction and are completely exposed in the tetramer recognized the peptide, the isolated subunit, and the oligomeric protein equally well. The responses of the T-cell lines and clones were major histocompatibility complex-restricted. Since the same x-irradiated antigen-presenting cells were employed, the results could not be attributed to differences or defects in Hb processing. The findings indicate that in vitro the native (unprocessed and undissociated) oligomeric protein was the trigger of major histocompatibility complex-restricted T-cell responses. PMID:2788890

  13. Design of a hyperstable 60-subunit protein icosahedron

    PubMed Central

    Hsia, Yang; Bale, Jacob B.; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K.; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N.; Gonen, Tamir; King, Neil P.; Baker, David

    2016-01-01

    The icosahedron and the dodecahedron are the largest of the Platonic solids, and icosahedral protein structures are widely utilized in biological systems for packaging and transport1,2. There has been considerable interest in repurposing such structures3–5, for example, virus-like particles for the targeted delivery and vaccine design. The ability to design proteins that self assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein 'containers' that could exhibit properties custom-made for various applications. In this manuscript, we describe the computational design of an icosahedral nano-cage that self-assembles from trimeric building blocks. Electron microscopy images of the designed protein expressed in E. coli reveals a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 M guanidine hydrochloride at up to 80 °C, and undergo extremely abrupt, but reversible, disassembly between 2 M and 2.25 M guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of superfolder GFP can be fused to each of the 60 subunits to create highly fluorescent standard candles for light microscopy, and a designed protein pentamer can be placed in the center of each of the twenty pentameric faces to potentially gate macromolecule access to the nanocage interior. Such robust designed nanocages should have considerable utility for targeted drug delivery6, vaccine design7, and synthetic biology8. PMID:27309817

  14. A small subunit processome protein promotes cancer by altering translation.

    PubMed

    Yang, H W; Kim, T-M; Song, S S; Menon, L; Jiang, X; Huang, W; Black, P M; Park, P J; Carroll, R S; Johnson, M D

    2015-08-20

    Dysregulation of ribosome biogenesis or translation can promote cancer, but the underlying mechanisms remain unclear. UTP18 is a component of the small subunit processome, a nucleolar multi-protein complex whose only known function is to cleave pre-ribosomal RNA to yield the 18S ribosomal RNA component of 40S ribosomal subunits. Here, we show that UTP18 also alters translation to promote stress resistance and growth, and that UTP18 is frequently gained and overexpressed in cancer. We observed that UTP18 localizes to the cytoplasm in a subset of cells, and that serum withdrawal increases cytoplasmic UTP18 localization. Cytoplasmic UTP18 associates with the translation complex and Hsp90 to upregulate the translation of IRES-containing transcripts such as HIF1a, Myc and VEGF, thereby inducing stress resistance. Hsp90 inhibition decreases cytoplasmic UTP18 and UTP18-induced increases in translation. Importantly, elevated UTP18 expression correlates with increased aggressiveness and decreased survival in numerous cancers. Enforced UTP18 overexpression promotes transformation and tumorigenesis, whereas UTP18 knockdown inhibits these processes. This stress adaptation mechanism is thus co-opted for growth by cancers, and its inhibition may represent a promising new therapeutic target.

  15. G alpha 12 and G alpha 13 subunits define a fourth class of G protein alpha subunits.

    PubMed Central

    Strathmann, M P; Simon, M I

    1991-01-01

    Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) are central to the signaling processes of multicellular organisms. We have explored the diversity of the G protein subunits in mammals and found evidence for a large family of genes that encode the alpha subunits. Amino acid sequence comparisons show that the different alpha subunits fall into at least three classes. These classes have been conserved in animals separated by considerable evolutionary distances; they are present in mammals, Drosophila, and nematodes. We have now obtained cDNA clones encoding two murine alpha subunits, G alpha 12 and G alpha 13, that define a fourth class. The translation products are predicted to have molecular masses of 44 kDa and to be insensitive to ADP-ribosylation by pertussis toxin. They share 67% amino acid sequence identity with each other and less than 45% identity with other alpha subunits. Their transcripts can be detected in every tissue examined, although the relative levels of the G alpha 13 message appear somewhat variable. Images PMID:1905812

  16. Flunitrazepam rapidly reduces GABAA receptor subunit protein expression via a protein kinase C-dependent mechanism

    PubMed Central

    Johnston, Jonathan D; Price, Sally A; Bristow, David R

    1998-01-01

    Acute flunitrazepam (1 μM) exposure for 1 h reduced GABAA receptor α1 (22±4%, mean±s.e.mean) and β2/3 (21±4%) subunit protein levels in cultured rat cerebellar granule cells. This rapid decrease in subunit proteins was completely prevented by bisindolymaleimide 1 (1 μM), an inhibitor of protein kinase C, but not by N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89, 4.8 μM), an inhibitor of protein kinases A and G. These results suggest the existence of a benzodiazepine-induced mechanism to rapidly alter GABAA receptor protein expression, that appears to be dependent on protein kinase C activity. PMID:9723942

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

    PubMed Central

    Asadabadi, Ebrahim Barzegari; Abdolmaleki, Parviz

    2013-01-01

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

  18. A revised model for AMP-activated protein kinase structure: The alpha-subunit binds to both the beta- and gamma-subunits although there is no direct binding between the beta- and gamma-subunits.

    PubMed

    Wong, Kelly A; Lodish, Harvey F

    2006-11-24

    The 5'-AMP-activated protein kinase (AMPK) is a master sensor for cellular metabolic energy state. It is activated by a high AMP/ATP ratio and leads to metabolic changes that conserve energy and utilize alternative cellular fuel sources. The kinase is composed of a heterotrimeric protein complex containing a catalytic alpha-subunit, an AMP-binding gamma-subunit, and a scaffolding beta-subunit thought to bind directly both the alpha- and gamma-subunits. Here, we use coimmunoprecipitation of proteins in transiently transfected cells to show that the alpha2-subunit binds directly not only to the beta-subunit, confirming previous work, but also to the gamma1-subunit. Deletion analysis of the alpha2-subunit reveals that the C-terminal 386-552 residues are sufficient to bind to the beta-subunit. The gamma1-subunit binds directly to the alpha2-subunit at two interaction sites, one within the catalytic domain consisting of alpha2 amino acids 1-312 and a second within residues 386-552. Binding of the alpha2 and the gamma1-subunits was not affected by 400 mum AMP or ATP. Furthermore, we show that the beta-subunit C terminus is essential for binding to the alpha2-subunit but, in contrast to previous work, the beta-subunit does not bind directly to the gamma1-subunit. Taken together, this study presents a new model for AMPK heterotrimer structure where through its C terminus the beta-subunit binds to the alpha-subunit that, in turn, binds to the gamma-subunit. There is no direct interaction between the beta- and gamma-subunits.

  19. Disrupting the Constitutive, Homodimeric Protein–Protein Interface in CK2β Using a Biophysical Fragment-Based Approach

    PubMed Central

    2016-01-01

    Identifying small molecules that induce the disruption of constitutive protein–protein interfaces is a challenging objective. Here, a targeted biophysical screening cascade was employed to specifically identify small molecules that could disrupt the constitutive, homodimeric protein–protein interface within CK2β. This approach could potentially be applied to achieve subunit disassembly of other homo-oligomeric proteins as a means of modulating protein function. PMID:27726344

  20. Interaction between G-protein beta and gamma subunit types is selective.

    PubMed Central

    Pronin, A N; Gautam, N

    1992-01-01

    Signal-transducing guanine nucleotide-binding proteins (G proteins) are made up of three subunits, alpha, beta, and gamma. Each of these subunits comprises a family of proteins. The rules for association between members of one family with members of another to form a multimer are not known; it is not clear whether associations are specific or nonspecific. Other than transducin (Gt), the G protein in rod photoreceptors, most purified G proteins contain more than one subtype of beta or gamma subunits. The Gt alpha subunit is associated only with beta 1 and gamma 1. It is not known whether this specificity is due to the differential expression of these subunit types in a cell type or due to intrinsically different affinities between different beta and gamma subunit types. We have used a transfected cell assay system to examine the association of the beta 1, beta 2, and beta 3 proteins with the gamma 1 and gamma 2 proteins. Results show that gamma 1 does not associate with beta 2 and that beta 3 does not associate with gamma 1 or gamma 2. Differences in affinities between types of G protein subunits will impose restrictions on the formation of certain heterotrimers and determine which G protein will be active in a cell. A chimeric molecule of beta 1 and beta 2 was used to broadly map the regions on these subunits that determine specificity of association. Images PMID:1631113

  1. The Protein-DNA Interface database

    PubMed Central

    2010-01-01

    The Protein-DNA Interface database (PDIdb) is a repository containing relevant structural information of Protein-DNA complexes solved by X-ray crystallography and available at the Protein Data Bank. The database includes a simple functional classification of the protein-DNA complexes that consists of three hierarchical levels: Class, Type and Subtype. This classification has been defined and manually curated by humans based on the information gathered from several sources that include PDB, PubMed, CATH, SCOP and COPS. The current version of the database contains only structures with resolution of 2.5 Å or higher, accounting for a total of 922 entries. The major aim of this database is to contribute to the understanding of the main rules that underlie the molecular recognition process between DNA and proteins. To this end, the database is focused on each specific atomic interface rather than on the separated binding partners. Therefore, each entry in this database consists of a single and independent protein-DNA interface. We hope that PDIdb will be useful to many researchers working in fields such as the prediction of transcription factor binding sites in DNA, the study of specificity determinants that mediate enzyme recognition events, engineering and design of new DNA binding proteins with distinct binding specificity and affinity, among others. Finally, due to its friendly and easy-to-use web interface, we hope that PDIdb will also serve educational and teaching purposes. PMID:20482798

  2. Metal templated design of protein interfaces.

    PubMed

    Salgado, Eric N; Ambroggio, Xavier I; Brodin, Jeffrey D; Lewis, Richard A; Kuhlman, Brian; Tezcan, F Akif

    2010-02-02

    Metal coordination is a key structural and functional component of a large fraction of proteins. Given this dual role we considered the possibility that metal coordination may have played a templating role in the early evolution of protein folds and complexes. We describe here a rational design approach, Metal Templated Interface Redesign (MeTIR), that mimics the time course of a hypothetical evolutionary pathway for the formation of stable protein assemblies through an initial metal coordination event. Using a folded monomeric protein, cytochrome cb(562), as a building block we show that its non-self-associating surface can be made self-associating through a minimal number of mutations that enable Zn coordination. The protein interfaces in the resulting Zn-directed, D(2)-symmetrical tetramer are subsequently redesigned, yielding unique protein architectures that self-assemble in the presence or absence of metals. Aside from its evolutionary implications, MeTIR provides a route to engineer de novo protein interfaces and metal coordination environments that can be tuned through the extensive noncovalent bonding interactions in these interfaces.

  3. Investigating the influence of ionic concentrations and subunit interactions on the self-assembly of E2 protein

    NASA Astrophysics Data System (ADS)

    Peng, Tao; Tan, Sze Wah; Dharmawan, Ratna Ekawati; Lim, Sierin

    2012-11-01

    Understanding of self-assembly mechanism of viruslike protein cage is important in controlling release of molecular cargo for applications in drug delivery. E2 core protein is composed of 60 subunits which self-assemble into a hollow 25-nm porous protein cage. Due to its virus like dodecahedral structure without infectious capacity, we are interested in its potential application as nanocapsule in drug delivery. In our study, extrinsic and intrinsic factors that influence self-assembly were evaluated. Extrinsic factors, such as salts and denaturants, were introduced into E2 protein solution. The hydrodynamic diameter of the E2 core protein was used to monitor its disassembly or aggregation. We found that the protein size increased proportionally with the salt concentration while the size decreased as the denaturant concentration increased. To assess intrinsic factors that influence E2 self-assembly, we identified some key amino acids at interfaces of subunits and performed site-directed mutagenesis on them. Characterizations of each mutant for size and secondary structure contents were performed. We found that mutations at the inner surface have no apparent effects on both protein sizes and secondary structures. The mutations at intra-trimer interfaces changed the secondary structure contents but the protein sizes remained stable.

  4. Single mutations at the subunit interface modulate copper reactivity in Photobacterium leiognathi Cu,Zn superoxide dismutase.

    PubMed

    Stroppolo, M E; Pesce, A; D'Orazio, M; O'Neill, P; Bordo, D; Rosano, C; Milani, M; Battistoni, A; Bolognesi, M; Desideri, A

    2001-05-04

    The functional properties and X-ray structures of five mutant forms of Photobacterium leiognathi Cu,Zn superoxide dismutase carrying single mutations at residues located at the dimer association interface have been investigated. When compared to the wild-type enzyme, the three-dimensional structures of the mutants show structural perturbations limited to the proximity of the mutation sites and substantial identity of active site geometry. Nonetheless, the catalytic rates of all mutants, measured at neutral pH and low ionic strength by pulse radiolysis, are higher than that of the wild-type protein. Such enzymatic activity increase is paralleled by enhanced active site accessibility to external chelating agents, which, in the mutated enzyme, remove more readily the active site copper ion. It is concluded that mutations at the prokaryotic Cu,Zn superoxide dismutase subunit interface can transduce dynamical perturbation to the active site region, promoting substrate active site accessibility. Such long-range intramolecular communication effects have not been extensively described before within the Cu,Zn superoxide dismutase homology family.

  5. Ric-8 Proteins Are Molecular Chaperones That Direct Nascent G Protein α Subunit Membrane Association

    PubMed Central

    Gabay, Meital; Pinter, Mary E.; Wright, Forrest A.; Chan, PuiYee; Murphy, Andrew J.; Valenzuela, David M.; Yancopoulos, George D.; Tall, Gregory G.

    2013-01-01

    Ric-8A (resistance to inhibitors of cholinesterase 8A) and Ric-8B are guanine nucleotide exchange factors that enhance different heterotrimeric guanine nucleotide–binding protein (G protein) signaling pathways by unknown mechanisms. Because transgenic disruption of Ric-8A or Ric-8B in mice caused early embryonic lethality, we derived viable Ric-8A– or Ric-8B–deleted embryonic stem (ES) cell lines from blastocysts of these mice. We observed pleiotropic G protein signaling defects in Ric-8A−/− ES cells, which resulted from reduced steady-state amounts of Gαi, Gαq, and Gα13 proteins to <5% of those of wild-type cells. The amounts of Gαs and total Gβ protein were partially reduced in Ric-8A−/− cells compared to those in wild-type cells, and only the amount of Gαs was reduced substantially in Ric-8B−/− cells. The abundances of mRNAs encoding the G protein α subunits were largely unchanged by loss of Ric-8A or Ric-8B. The plasma membrane residence of G proteins persisted in the absence of Ric-8 but was markedly reduced compared to that in wild-type cells. Endogenous Gαi and Gαq were efficiently translated in Ric-8A−/− cells but integrated into endomembranes poorly; however, the reduced amounts of G protein α subunits that reached the membrane still bound to nascent Gβγ. Finally, Gαi, Gαq, and Gβ1 proteins exhibited accelerated rates of degradation in Ric-8A−/− cells compared to those in wild-type cells. Together, these data suggest that Ric-8 proteins are molecular chaperones required for the initial association of nascent Gα subunits with cellular membranes. PMID:22114146

  6. Differential expression of the protein kinase A subunits in normal adrenal glands and adrenocortical adenomas.

    PubMed

    Weigand, Isabel; Ronchi, Cristina L; Rizk-Rabin, Marthe; Dalmazi, Guido Di; Wild, Vanessa; Bathon, Kerstin; Rubin, Beatrice; Calebiro, Davide; Beuschlein, Felix; Bertherat, Jérôme; Fassnacht, Martin; Sbiera, Silviu

    2017-12-01

    Somatic mutations in protein kinase A catalytic α subunit (PRKACA) were found to be causative for 30-40% of cortisol-producing adenomas (CPA) of the adrenal gland, rendering PKA signalling constitutively active. In its resting state, PKA is a stable and inactive heterotetramer, consisting of two catalytic and two regulatory subunits with the latter inhibiting PKA activity. The human genome encodes three different PKA catalytic subunits and four different regulatory subunits that are preferentially expressed in different organs. In normal adrenal glands all regulatory subunits are expressed, while CPA exhibit reduced protein levels of the regulatory subunit IIβ. In this study, we linked for the first time the loss of RIIβ protein levels to the PRKACA mutation status and found the down-regulation of RIIβ to arise post-transcriptionally. We further found the PKA subunit expression pattern of different tumours is also present in the zones of the normal adrenal cortex and demonstrate that the different PKA subunits have a differential expression pattern in each zone of the normal adrenal gland, indicating potential specific roles of these subunits in the regulation of different hormones secretion.

  7. Determination of kainate receptor subunit ratios in mouse brain using novel chimeric protein standards.

    PubMed

    Watanabe-Iida, Izumi; Konno, Kohtarou; Akashi, Kaori; Abe, Manabu; Natsume, Rie; Watanabe, Masahiko; Sakimura, Kenji

    2016-01-01

    Kainate-type glutamate receptors (KARs) are tetrameric channels assembled from GluK1-5. GluK1-3 are low-affinity subunits that form homomeric and heteromeric KARs, while GluK4 and GluK5 are high-affinity subunits that require co-assembly with GluK1-3 for functional expression. Although the subunit composition is thought to be highly heterogeneous in the brain, the distribution of KAR subunits at the protein level and their relative abundance in given regions of the brain remain largely unknown. In the present study, we titrated C-terminal antibodies to each KAR subunit using chimeric GluA2-GluK fusion proteins, and measured their relative abundance in the P2 and post-synaptic density (PSD) fractions of the adult mouse hippocampus and cerebellum. Analytical western blots showed that GluK2 and GluK3 were the major KAR subunits, with additional expression of GluK5 in the hippocampus and cerebellum. In both regions, GluK4 was very low and GluK1 was below the detection threshold. The relative amount of low-affinity subunits (GluK2 plus GluK3) was several times higher than that of high-affinity subunits (GluK4 plus GluK5) in both regions. Of note, the highest ratio of high-affinity subunits to low-affinity subunits was found in the hippocampal PSD fraction (0.32), suggesting that heteromeric receptors consisting of high- and low-affinity subunits highly accumulate at hippocampal synapses. In comparison, this ratio was decreased to 0.15 in the cerebellar PSD fraction, suggesting that KARs consisting of low-affinity subunits are more prevalent in the cerebellum. Therefore, low-affinity KAR subunits are predominant in the brain, with distinct subunit combinations between the hippocampus and cerebellum. Kainate receptors, an unconventional member of the iGluR receptor family, have a tetrameric structure assembled from low-affinity (GluK1-3) and high-affinity (GluK4 and GluK5) subunits. We used a simple but novel procedure to measure the relative abundance of both low- and

  8. Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site

    PubMed Central

    Wongsantichon, Jantana; Robinson, Robert C.; Ketterman, Albert J.

    2015-01-01

    Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme. PMID:26487708

  9. Evolvability of yeast protein-protein interaction interfaces.

    PubMed

    Talavera, David; Williams, Simon G; Norris, Matthew G S; Robertson, David L; Lovell, Simon C

    2012-06-22

    The functional importance of protein-protein interactions indicates that there should be strong evolutionary constraint on their interaction interfaces. However, binding interfaces are frequently affected by amino acid replacements. Change due to coevolution within interfaces can contribute to variability but is not ubiquitous. An alternative explanation for the ability of surfaces to accept replacements may be that many residues can be changed without affecting the interaction. Candidates for these types of residues are those that make interchain interaction only through the protein main chain, β-carbon, or associated hydrogen atoms. Since almost all residues have these atoms, we hypothesize that this subset of interface residues may be more easily substituted than those that make interactions through other atoms. We term such interactions "residue type independent." Investigating this hypothesis, we find that nearly a quarter of residues in protein interaction interfaces make exclusively interchain residue-type-independent contacts. These residues are less structurally constrained and less conserved than residues making residue-type-specific interactions. We propose that residue-type-independent interactions allow substitutions in binding interfaces while the specificity of binding is maintained.

  10. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development

    PubMed Central

    Beier, Anna; Krisp, Christoph; Wolters, Dirk A.

    2016-01-01

    ABSTRACT The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora. Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. PMID:27329756

  11. Proteins at the Biomaterial Electrolyte Interface

    NASA Astrophysics Data System (ADS)

    Tengvall, Pentti

    2005-03-01

    Proteins adsorb rapidly onto solid and polymeric surfaces because the association process is in the vast majority of cases energetically favourable, i.e. exothermic. The most common exceptions to this rule are hydrophilic interfaces with low net charge and high mobility, e.g. immobilized PEGs. Current research in the research area tries to understand and control unwanted and wanted adsorption by studying the adsorption kinetics, protein surface binding specificity, protein exchange at interfaces, and surface protein repulsion mechanisms. In blood plasma model systems humoral cascade reactions such as surface mediated coagulation and immune complement raise considerable interest due to the immediate association to blood compatibility, and in tissue applications the binding between surfaces and membrane receptors in cells and tissues. Thus, the understanding of interfacial events at the protein level is of large importance in applications such as blood and tissue contacting biomaterials, in vitro medical and biological diagnostics, food industry and in marine anti-fouling technology. Well described consequences of adsorption are a lowered system energy, increased system entropy, irreversible binding, conformational changes, specific surface/protein interactions, and in biomedical materials applications surface opsonization followed by cell-surface interactions and a host tissue response. This lecture will deal with some mechanisms known to be of importance for the adsorption processes, such as the influence of surface chemistry and surface energy, the composition of the protein solution, the Vroman effect, and residence time. Examples will be shown from ellipsometric experiments using different model surfaces in single/few protein solutions, and specific attention be given to blood serum and plasma experiments on coagulation and immune complement at interfaces.

  12. The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes.

    PubMed Central

    Solomon, K R; Rudd, C E; Finberg, R W

    1996-01-01

    Glycosylphosphatidylinositol (GPI)-anchored proteins are nonmembrane spanning cell surface proteins that have been demonstrated to be signal transduction molecules. Because these proteins do not extend into the cytoplasm, the mechanism by which cross-linking of these molecules leads to intracellular signal transduction events is obscure. Previous analysis has indicated that these proteins are associated with src family member tyrosine kinases; however, the role this interaction plays in the generation of intracellular signals is not clear. Here we show that GPI-anchored proteins are associated with alpha subunits of heterotrimeric GTP binding proteins (G proteins) in both human and murine lymphocytes. When the GPI-anchored proteins CD59, CD48, and Thy-1 were immunoprecipitated from various cell lines or freshly isolated lymphocytes, all were found to be associated with a 41-kDa phosphoprotein that we have identified, by using specific antisera, as a mixture of tyrosine phosphorylated G protein alpha subunits: a small amount of Gialpha1, and substantial amounts of Gialpha2 and Gialpha3. GTP binding assays performed with immunoprecipitations of CD59 indicated that there was GTP-binding activity associated with this molecule. Thus, we have shown by both immunochemical and functional criteria that GPI-anchored proteins are physically associated with G proteins. These experiments suggest a potential role of G proteins in the transduction of signals generated by GPI-anchored molecules expressed on lymphocytes of both mouse and human. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8650218

  13. Characterization of the interface between gamma and epsilon subunits of Escherichia coli F1-ATPase.

    PubMed

    Tang, C; Capaldi, R A

    1996-02-09

    The interaction faces of the gamma and epsilon subunits in the Escherichia coli F1-ATPase have been explored by a combination of cross-linking and chemical modification experiments using several mutant epsilon subunits as follows: epsilonS10C, epsilonH38C, epsilonT43C, epsilonS65C, epsilonS108C, and epsilonM138C, along with a mutant of the gamma subunit, gammaT106C. The replacement of Ser-10 by a Cys or Met-138 by a Cys reduced the inhibition of ECF1 by the epsilon subunit, while the mutation S65C increased this inhibitory effect. Modification of the Cys at position 10 with N-ethylmaleimide or fluoroscein maleimide further reduced the binding affinity of, and the maximal inhibition by, the epsilon subunit. Similar chemical modification of the Cys at position 43 of the epsilon subunit (in the mutant epsilonT43C) and a Cys at position 106 of the gamma subunit (gammaT106C) also affected the inhibition of ECF1 by the epsilon subunit. The various epsilon subunit mutants were reacted with TFPAM3, and the site(s) of cross-linking within the ECF1 complex was determined. Previous studies have shown cross-linking from the Cys at positions 10 and 38 with the gamma subunit and from a Cys at position 108 to an alpha subunit (Aggeler, R., Chicas-Cruz, K., Cai, S. X., Keana, J. F. W., and Capaldi, R. A. (1992) Biochemistry 31, 2956-2961; Aggeler, R., Weinreich, F., and Capaldi, R. A. (1995) Biochim. Biophys. Acta 1230, 62-68). Here, cross-linking was found from a Cys at position 43 to the gamma subunit and from the Cys at position 138 to a beta subunit. The site of cross-linking from Cys-10 of epsilon to the gamma subunit was localized by peptide mapping to a region of the gamma subunit between residues 222 and 242. Cross-linking from a Cys at position 38 and at position 43 was with the C-terminal part of the gamma subunit, between residues 202 and 286. ECF1 treated with trypsin at pH 7.0 still binds purified epsilon subunit, while enzyme treated with the protease at pH 8.0 does

  14. Interface-Resolved Network of Protein-Protein Interactions

    PubMed Central

    Johnson, Margaret E.; Hummer, Gerhard

    2013-01-01

    We define an interface-interaction network (IIN) to capture the specificity and competition between protein-protein interactions (PPI). This new type of network represents interactions between individual interfaces used in functional protein binding and thereby contains the detail necessary to describe the competition and cooperation between any pair of binding partners. Here we establish a general framework for the construction of IINs that merges computational structure-based interface assignment with careful curation of available literature. To complement limited structural data, the inclusion of biochemical data is critical for achieving the accuracy and completeness necessary to analyze the specificity and competition between the protein interactions. Firstly, this procedure provides a means to clarify the information content of existing data on purported protein interactions and to remove indirect and spurious interactions. Secondly, the IIN we have constructed here for proteins involved in clathrin-mediated endocytosis (CME) exhibits distinctive topological properties. In contrast to PPI networks with their global and relatively dense connectivity, the fragmentation of the IIN into distinctive network modules suggests that different functional pressures act on the evolution of its topology. Large modules in the IIN are formed by interfaces sharing specificity for certain domain types, such as SH3 domains distributed across different proteins. The shared and distinct specificity of an interface is necessary for effective negative and positive design of highly selective binding targets. Lastly, the organization of detailed structural data in a network format allows one to identify pathways of specific binding interactions and thereby predict effects of mutations at specific surfaces on a protein and of specific binding inhibitors, as we explore in several examples. Overall, the endocytosis IIN is remarkably complex and rich in features masked in the coarser

  15. Studies on the murine Ss protein. I. Purification, molecular weight, and subunit structure

    PubMed Central

    1975-01-01

    The murine Ss protein has been isolated and purified. Using specific antisera, the radiolabeled protein has a mol wt of 120,000 in sodium dodecyl sulfate polyacrylamide gels. It is composed of two basic subunits of 23,000 and 14,000 daltons. The smaller molecular weight subunit contains a single disulfide bridge, is devoid of carbohydrate, and may represent the murine equivalent of beta2-microglobulin. PMID:809530

  16. Cation-pi interactions in protein-protein interfaces.

    PubMed

    Crowley, Peter B; Golovin, Adel

    2005-05-01

    Arginine is an abundant residue in protein-protein interfaces. The importance of this residue relates to the versatility of its side chain in intermolecular interactions. Different classes of protein-protein interfaces were surveyed for cation-pi interactions. Approximately half of the protein complexes and one-third of the homodimers analyzed were found to contain at least one intermolecular cation-pi pair. Interactions between arginine and tyrosine were found to be the most abundant. The electrostatic interaction energy was calculated to be approximately 3 kcal/mol, on average. A distance-based search of guanidinium:aromatic interactions was also performed using the Macromolecular Structure Database (MSD). This search revealed that half of the guanidinium:aromatic pairs pack in a coplanar manner. Furthermore, it was found that the cationic group of the cation-pi pair is frequently involved in intermolecular hydrogen bonds. In this manner the arginine side chain can participate in multiple interactions, providing a mechanism for inter-protein specificity. Thus, the cation-pi interaction is established as an important contributor to protein-protein interfaces.

  17. Influence of gamma subunit prenylation on association of guanine nucleotide-binding regulatory proteins with membranes.

    PubMed Central

    Muntz, K H; Sternweis, P C; Gilman, A G; Mumby, S M

    1992-01-01

    Two approaches were taken to address the possible role of gamma-subunit prenylation in dictating the cellular distribution of guanine nucleotide-binding regulatory proteins. Prenylation of gamma subunits was prevented by site-directed mutagenesis or by inhibiting the synthesis of mevalonate, the precursor of cellular isoprenoids. When beta or gamma subunits were transiently expressed in COS-M6 simian kidney cells (COS) cells, the proteins were found in the membrane fraction by immunoblotting. Immunofluorescence experiments indicated that the proteins were distributed to intracellular structures in addition to plasma membranes. Replacement of Cys68 of gamma with Ser prevented prenylation of the mutant protein and association of the protein with the membrane fraction of COS cells. Immunoblotting results demonstrated that some of the beta subunits were found in the cytoplasm when coexpressed with the nonprenylated mutant gamma subunit. When Neuro 2A cells were treated with compactin to inhibit protein prenylation, a fraction of endogenous beta and gamma was distributed in the cytoplasm. It is concluded that prenylation facilitates association of gamma subunits with membranes, that the cellular location of gamma influences the distribution of beta, and that prenylation is not an absolute requirement for interaction of beta and gamma. Images PMID:1550955

  18. 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

    2017-01-27

    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.

  19. Specific Inhibition of Herpes Simplex Virus DNA Polymerase by Helical Peptides Corresponding to the Subunit Interface

    NASA Astrophysics Data System (ADS)

    Digard, Paul; Williams, Kevin P.; Hensley, Preston; Brooks, Ian S.; Dahl, Charles E.; Coen, Donald M.

    1995-02-01

    The herpes simplex virus DNA polymerase consists of two subunits-a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial α-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of α-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase δ and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.

  20. Membrane insertion and topology of the translocon-associated protein (TRAP) gamma subunit.

    PubMed

    Bañó-Polo, Manuel; Martínez-Garay, Carlos A; Grau, Brayan; Martínez-Gil, Luis; Mingarro, Ismael

    2017-05-01

    Translocon-associated protein (TRAP) complex is intimately associated with the ER translocon for the insertion or translocation of newly synthesised proteins in eukaryotic cells. The TRAP complex is comprised of three single-spanning and one multiple-spanning subunits. We have investigated the membrane insertion and topology of the multiple-spanning TRAP-γ subunit by glycosylation mapping and green fluorescent protein fusions both in vitro and in cell cultures. Results demonstrate that TRAP-γ has four transmembrane (TM) segments, an Nt/Ct cytosolic orientation and that the less hydrophobic TM segment inserts efficiently into the membrane only in the cellular context of full-length protein.

  1. Deciphering the subunit composition of multimeric proteins by counting photobleaching steps.

    PubMed

    Arant, Ryan J; Ulbrich, Maximilian H

    2014-03-17

    The limit of subdiffraction imaging with fluorescent proteins currently lies at 20 nm, and therefore most protein complexes are too small (2-5 nm) to spatially resolve their individual subunits by optical means. However, the number and stoichiometry of subunits within an immobilized protein complex can be resolved by the observation of photobleaching steps of individual fluorophores or co-localization of single-molecule fluorescence emission in multiple colors. We give an overview of the proteins that have been investigated by this approach and the different techniques that can be used to immobilize and label the proteins. This minireview should serve as a guideline for scientists who want to employ single-molecule subunit counting for their research.

  2. Structure-based design of small peptide inhibitors of protein kinase CK2 subunit interaction

    PubMed Central

    Laudet, Béatrice; Barette, Caroline; Dulery, Vincent; Renaudet, Olivier; Dumy, Pascal; Metz, Alexandra; Prudent, Renaud; Deshiere, Alexandre; Dideberg, Otto; Filhol, Odile; Cochet, Claude

    2007-01-01

    X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged the traditional view of protein kinase CK2. Unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumours. Thus the potential intersubunit flexibility suggested by these studies raises the likely prospect that the CK2 holoenzyme complex is subject to disassembly and reassembly. In the present paper, we show evidence for the reversible multimeric organization of the CK2 holoenzyme complex in vitro. We used a combination of site-directed mutagenesis, binding experiments and functional assays to show that, both in vitro and in vivo, only a small set of primary hydrophobic residues of CK2β which contacts at the centre of the CK2α/CK2β interface dominates affinity. The results indicate that a double mutation in CK2β of amino acids Tyr188 and Phe190, which are complementary and fill up a hydrophobic pocket of CK2α, is the most disruptive to CK2α binding both in vitro and in living cells. Further characterization of hotspots in a cluster of hydrophobic amino acids centred around Tyr188–Phe190 led us to the structure-based design of small-peptide inhibitors. One conformationally constrained 11-mer peptide (Pc) represents a unique CK2β-based small molecule that was particularly efficient (i) to antagonize the interaction between the CK2 subunits, (ii) to inhibit the assembly of the CK2 holoenzyme complex, and (iii) to strongly affect its substrate preference. PMID:17714077

  3. Type B Heterotrimeric G Protein γ-Subunit Regulates Auxin and ABA Signaling in Tomato.

    PubMed

    Subramaniam, Gayathery; Trusov, Yuri; Lopez-Encina, Carlos; Hayashi, Satomi; Batley, Jacqueline; Botella, José Ramón

    2016-02-01

    Heterotrimeric G proteins composed of α, β, and γ subunits are central signal transducers mediating the cellular response to multiple stimuli in most eukaryotes. Gγ subunits provide proper cellular localization and functional specificity to the heterotrimer complex. Plant Gγ subunits, divided into three structurally distinct types, are more diverse than their animal counterparts. Type B Gγ subunits, lacking a carboxyl-terminal isoprenylation motif, are found only in flowering plants. We present the functional characterization of type B Gγ subunit (SlGGB1) in tomato (Solanum lycopersicum). We show that SlGGB1 is the most abundant Gγ subunit in tomato and strongly interacts with the Gβ subunit. Importantly, the green fluorescent protein-SlGGB1 fusion protein as well as the carboxyl-terminal yellow fluorescent protein-SlGGB1/amino-terminal yellow fluorescent protein-Gβ heterodimer were localized in the plasma membrane, nucleus, and cytoplasm. RNA interference-mediated silencing of SlGGB1 resulted in smaller seeds, higher number of lateral roots, and pointy fruits. The silenced lines were hypersensitive to exogenous auxin, while levels of endogenous auxins were lower or similar to those of the wild type. SlGGB1-silenced plants also showed strong hyposensitivity to abscisic acid (ABA) during seed germination but not in other related assays. Transcriptome analysis of the transgenic seeds revealed abnormal expression of genes involved in ABA sensing, signaling, and response. We conclude that the type B Gγ subunit SlGGB1 mediates auxin and ABA signaling in tomato.

  4. Assembly and Intracellular Targeting of the βγ Subunits of Heterotrimeric G Proteins

    PubMed Central

    Rehm, Armin; Ploegh, Hidde L.

    1997-01-01

    The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent α, β, and γ subunits is a complex process, compounded by the multiplicity of the genes that encode them, and the diversity of receptors and effectors with which they interact. Monoclonal anti-β antibodies (ARC5 and ARC9), raised against immunoaffinity purified βγ complexes, recognize β subunits when not associated with γ and can thus be used to monitor assembly of βγ complexes. Complex formation starts immediately after synthesis and is complete within 30 min. Assembly occurs predominantly in the cytosol, and association of βγ complexes with the plasma membrane fraction starts between 15–30 min of chase. Three pools of β subunits can be distinguished based on their association with γ subunits, their localization, and their detergent solubility. Association of β and α subunits with detergent-insoluble domains occurs within 1 min of chase, and increases to reach a plateau of near complete detergent resistance within 30 min of chase. Brefeldin A treatment does not interfere with delivery of βγ subunits to detergent-insoluble domains, suggesting that assembly of G protein subunits with their receptors occurs distally from the BFA-imposed block of intracellular membrane trafficking and may occur directly at the plasma membrane. PMID:9128244

  5. The Interface between Membrane-Spanning and Cytosolic Domains in Ca2+-Dependent K+ Channels Is Involved in β Subunit Modulation of Gating

    PubMed Central

    Sun, Xiaohui; Shi, Jingyi; Delaloye, Kelli; Yang, Xiao; Yang, Huanghe; Zhang, Guohui

    2013-01-01

    Large-conductance, voltage-, and Ca2+-dependent K+ (BK) channels are broadly expressed in various tissues to modulate neuronal activity, smooth muscle contraction, and secretion. BK channel activation depends on the interactions among the voltage sensing domain (VSD), the cytosolic domain (CTD), and the pore gate domain (PGD) of the Slo1 α-subunit, and is further regulated by accessory β subunits (β1–β4). How β subunits fine-tune BK channel activation is critical to understand the tissue-specific functions of BK channels. Multiple sites in both Slo1 and the β subunits have been identified to contribute to the interaction between Slo1 and the β subunits. However, it is unclear whether and how the interdomain interactions among the VSD, CTD, and PGD are altered by the β subunits to affect channel activation. Here we show that human β1 and β2 subunits alter interactions between bound Mg2+ and gating charge R213 and disrupt the disulfide bond formation at the VSD–CTD interface of mouse Slo1, indicating that the β subunits alter the VSD–CTD interface. Reciprocally, mutations in the Slo1 that alter the VSD–CTD interaction can specifically change the effects of the β1 subunit on the Ca2+ activation and of the β2 subunit on the voltage activation. Together, our data suggest a novel mechanism by which the β subunits modulated BK channel activation such that a β subunit may interact with the VSD or the CTD and alter the VSD–CTD interface of the Slo1, which enables the β subunit to have effects broadly on both voltage and Ca2+-dependent activation. PMID:23825428

  6. Differential distribution of G-protein beta-subunits in brain: an immunocytochemical analysis.

    PubMed

    Brunk, I; Pahner, I; Maier, U; Jenner, B; Veh, R W; Nürnberg, B; Ahnert-Hilger, G

    1999-05-01

    Heterotrimeric G proteins play central roles in signal transduction of neurons and other cells. The variety of their alpha-, beta-, and gamma-subunits allows numerous combinations thereby confering specificity to receptor-G-protein-effector interactions. Using antisera against individual G-protein beta-subunits we here present a regional and subcellular distribution of Gbeta1, Gbeta2, and Gbeta5 in rat brain. Immunocytochemical specificity of the subtype-specific antisera is revealed in Sf9 cells infected with various G-protein beta-subunits. Since Gbeta-subunits together with a G-protein gamma-subunit affect signal cascades we include a distribution of the neuron-specific Ggamma2- and Ggamma3-subunits in selected brain areas. Gbeta1, Gbeta2, and Gbeta5 are preferentially distributed in the neuropil of hippocampus, cerebellum and spinal cord. Gbeta2 is highly concentrated in the mossy fibres of dentate gyrus neurons ending in the stratum lucidum of hippocampal CA3-area. High amounts of Gbeta2 also occur in interneurons innervating spinal cord alpha-motoneurons. Gbeta5 is differentially distributed in all brain areas studied. It is found in the pyramidal cells of hippocampal CA1-CA3 as well as in the granule cell layer of dentate gyrus and in some interneurons. In the spinal cord Gbeta5 in contrast to Gbeta2 concentrates around alpha-motoneurons. In cultivated mouse hippocampal and hypothalamic neurons Gbeta2 and Gbeta5 are found in different subcellular compartments. Whereas Gbeta5 is restricted to the perikarya, Gbeta2 is also found in processes and synaptic contacts where it partially colocalizes with the synaptic vesicle protein synaptobrevin. An antiserum recognizing Ggamma2 and Ggamma3 reveals that these subunits are less expressed in hippocampus and cerebellum. Presumably this antiserum specifically recognizes Ggamma2 and Ggamma3 in combinations with certain G alphas and/or Gbetas. The widespread but regionally and cellularly rather different distribution of

  7. Implication of Terminal Residues at Protein-Protein and Protein-DNA Interfaces.

    PubMed

    Martin, Olivier M F; Etheve, Loïc; Launay, Guillaume; Martin, Juliette

    2016-01-01

    Terminal residues of protein chains are charged and more flexible than other residues since they are constrained only on one side. Do they play a particular role in protein-protein and protein-DNA interfaces? To answer this question, we considered large sets of non-redundant protein-protein and protein-DNA complexes and analyzed the status of terminal residues and their involvement in interfaces. In protein-protein complexes, we found that more than half of terminal residues (62%) are either modified by attachment of a tag peptide (10%) or have missing coordinates in the analyzed structures (52%). Terminal residues are almost exclusively located at the surface of proteins (94%). Contrary to charged residues, they are not over or under-represented in protein-protein interfaces, but strongly prefer the peripheral region of interfaces when present at the interface (83% of terminal residues). The almost exclusive location of terminal residues at the surface of the proteins or in the rim regions of interfaces explains that experimental methods relying on tail hybridization can be successfully applied without disrupting the complexes under study. Concerning conformational rearrangement in protein-protein complexes, despite their expected flexibility, terminal residues adopt similar locations between the free and bound forms of the docking benchmark. In protein-DNA complexes, N-terminal residues are twice more frequent than C-terminal residues at interfaces. Both N-terminal and C-terminal residues are under-represented in interfaces, in contrast to positively charged residues, which are strongly favored. When located in protein-DNA interfaces, terminal residues prefer the periphery. N-terminal and C-terminal residues thus have particular properties with regard to interfaces, which cannot be reduced to their charged nature.

  8. A hybrid NMR/SAXS-based approach for discriminating oligomeric protein interfaces using Rosetta

    PubMed Central

    Rossi, Paolo; Shi, Lei; Liu, Gaohua; Barbieri, Christopher M.; Lee, Hsiau-Wei; Grant, Thomas D.; Luft, Joseph R.; Xiao, Rong; Acton, Thomas B.; Snell, Edward H.; Montelione, Gaetano T.; Baker, David; Lange, Oliver F.; Sgourakis, Nikolaos G.

    2016-01-01

    Oligomeric proteins are important targets for structure determination in solution. While in most cases the fold of individual subunits can be determined experimentally, or predicted by homology-based methods, protein-protein interfaces are challenging to determine de novo using conventional NMR structure determination protocols. Here we focus on a member of the bet-V1 superfamily, Aha1 from Colwellia psychrerythraea. This family displays a broad range of crystallographic interfaces none of which can be reconciled with the NMR and SAXS data collected for Aha1. Unlike conventional methods relying on a dense network of experimental restraints, the sparse data are used to limit conformational search during optimization of a physically realistic energy function. This work highlights a new approach for studying minor conformational changes due to structural plasticity within a single dimeric interface in solution. PMID:25388768

  9. Fast-channel congenital myasthenic syndrome with a novel acetylcholine receptor mutation at the α-ε subunit interface.

    PubMed

    Webster, Richard; Liu, Wei-Wei; Chaouch, Amina; Lochmüller, Hanns; Beeson, David

    2014-02-01

    Congenital myasthenic syndromes (CMS) result from the failure to achieve muscle depolarisation due to disorders in the structure and/or function of the neuromuscular synapse. Mutations of the nicotinic acetylcholine receptor (nAChR) form a major subset of CMS. We describe a patient who presented with recurrent apnoeic crises in the neonatal period requiring ventilator support. Electromyography revealed compound muscle action potential decrement upon repetitive stimulation. Sequencing of nAChR subunit genes revealed two missense mutations. One previously reported null mutation p.εTyr15His, and a second novel missense mutation, p.εThr38Lys, that is well expressed in mammalian cell culture and thus likely to exert its effect via alteration of ion channel kinetics. Functional analysis revealed abbreviated ion channel bursts characteristic of a fast channel CMS. The mutation p.εThr38Lys occurs at the interface between the α and ε subunits of the nAChR pentamer and leads to instability of the open channel. The effects of this mutation on channel function were investigated in relation to other fast channel mutants at an analogous subunit interface within the nAChR pentamer. Fast channel syndromes are frequently characterised by severe myasthenic weakness with apnoeic crises; knowledge of the underlying mutation and its functional consequences can be vital for appropriate therapy and patient management.

  10. Understanding Molecular Recognition by G protein βγ Subunits on the Path to Pharmacological Targeting

    PubMed Central

    Lin, Yuan

    2011-01-01

    Heterotrimeric G proteins, composed of Gα and Gβγ subunits, transduce extracellular signals via G-protein-coupled receptors to modulate many important intracellular responses. The Gβγ subunits hold a central position in this signaling system and have been implicated in multiple aspects of physiology and the pathophysiology of disease. The Gβ subunit belongs to a large family of WD40 repeat proteins with a circular β-bladed propeller structure. This structure allows Gβγ to interact with a broad range of proteins to play diverse roles. How Gβγ interacts with and regulates such a wide variety of partners yet maintains specificity is an interesting problem in protein-protein molecular recognition in signal transduction, where signal transfer by proteins is often driven by modular conserved recognition motifs. Evidence has accumulated that one mechanism for Gβγ multitarget recognition is through an intrinsically flexible protein surface or “hot spot” that accommodates multiple modes of binding. Because each target has a unique recognition mode for Gβγ subunits, it suggests that these interactions could be selectively manipulated with small molecules, which could have significant therapeutic potential. PMID:21737569

  11. Mycobacterial proteins--immune targets for antituberculous subunit vaccine.

    PubMed

    Dhiman, N; Khuller, G K

    1999-12-01

    Cellular and humoral immunity induced by Mycobacterium tuberculosis has led to identification of newer vaccine candidates, but despite this, many questions concerning the protection against tuberculosis remain unanswered. Recent progress in this field has centered on T cell subset responses and cytokines that these cells secrete. There has been a steady progress in identification and characterization of several classes of major mycobacterial proteins which includes secretory/export proteins, cell wall associated proteins, heat shock proteins and cytoplasmic proteins. The protein antigens are now believed to represent the key protective immunity inducing antigens in the bacillus. In this review, various mycobacterial protein antigens of vaccination potential are compared for their efficacy in light of current immunological knowledge.

  12. Eukaryote-specific extensions in ribosomal proteins of the small subunit: Structure and function

    PubMed Central

    Ghosh, Arnab; Komar, Anton A

    2015-01-01

    High-resolution structures of yeast ribosomes have improved our understanding of the architecture and organization of eukaryotic rRNA and proteins, as well as eukaryote-specific extensions present in some conserved ribosomal proteins. Despite this progress, assignment of specific functions to individual proteins and/or eukaryote-specific protein extensions remains challenging. It has been suggested that eukaryote-specific extensions of conserved proteins from the small ribosomal subunit may facilitate eukaryote-specific reactions in the initiation phase of protein synthesis. This review summarizes emerging data describing the structural and functional significance of eukaryote-specific extensions of conserved small ribosomal subunit proteins, particularly their possible roles in recruitment and spatial organization of eukaryote-specific initiation factors. PMID:26779416

  13. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human

    SciTech Connect

    Blatt, C.; Eversole-Cire, P.; Cohn, V.H.; Zollman, S.; Fournier, R.E.K.; Mohandas, L.T.; Nesbitt, M.; Lugo, T.; Jones, D.T.; Reed, R.R.; Weiner, L.P.; Sparkes, R.S.; Simon, M.I. )

    1988-10-01

    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding {alpha}-subunit proteins, two different {beta} subunits, and one {gamma} subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The {beta} subunits were also assigned-GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extend of the G{alpha} gene family and may help in attempts to correlate specific genetic diseases and with genes corresponding to G proteins.

  14. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

    PubMed Central

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

    2014-01-01

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

  15. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex.

    PubMed

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B; Webb, Kristofor; Bennett, Eric J; Vinterbo, Staal; Potter, Clinton S; Carragher, Bridget; Joazeiro, Claudio A P

    2014-11-11

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes.

  16. Pathogen mimicry of host protein-protein interfaces modulates immunity.

    PubMed

    Guven-Maiorov, Emine; Tsai, Chung-Jung; Nussinov, Ruth

    2016-10-01

    Signaling pathways shape and transmit the cell's reaction to its changing environment; however, pathogens can circumvent this response by manipulating host signaling. To subvert host defense, they beat it at its own game: they hijack host pathways by mimicking the binding surfaces of host-encoded proteins. For this, it is not necessary to achieve global protein homology; imitating merely the interaction surface is sufficient. Different protein folds often interact via similar protein-protein interface architectures. This similarity in binding surfaces permits the pathogenic protein to compete with a host target protein. Thus, rather than binding a host-encoded partner, the host protein hub binds the pathogenic surrogate. The outcome can be dire: rewiring or repurposing the host pathways, shifting the cell signaling landscape and consequently the immune response. They can also cause persistent infections as well as cancer by modulating key signaling pathways, such as those involving Ras. Mapping the rewired host-pathogen 'superorganism' interaction network - along with its structural details - is critical for in-depth understanding of pathogenic mechanisms and developing efficient therapeutics. Here, we overview the role of molecular mimicry in pathogen host evasion as well as types of molecular mimicry mechanisms that emerged during evolution.

  17. Molecular mechanism of activation-triggered subunit exchange in Ca2+/calmodulin-dependent protein kinase II

    PubMed Central

    Bhattacharyya, Moitrayee; Stratton, Margaret M; Going, Catherine C; McSpadden, Ethan D; Huang, Yongjian; Susa, Anna C; Elleman, Anna; Cao, Yumeng Melody; Pappireddi, Nishant; Burkhardt, Pawel; Gee, Christine L; Barros, Tiago; Schulman, Howard; Williams, Evan R; Kuriyan, John

    2016-01-01

    Activation triggers the exchange of subunits in Ca2+/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This converts the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones. DOI: http://dx.doi.org/10.7554/eLife.13405.001 PMID:26949248

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

    PubMed

    Tuncbag, Nurcan; Gursoy, Attila; Keskin, Ozlem

    2011-06-01

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

  19. Inheritance behavior of information coding for small subunit polypeptides of fraction 1 protein.

    PubMed

    Chen, K; Wildman, S G

    1980-12-01

    In various genera of plants, the small subunit of fraction 1 protein is often composed of more than one kind of polypeptide; these differ in isoelectric points and amino acid composition. Previous analysis of numerous individual progeny of Nicotiana tabacum (two kinds of polypeptides), N. glauca + N. langsdorffii parasexual hybrids (three kinds) and other examples showed no change in F-1 protein composition as a consequence of alternation of generations. Experiments reported here show that absence of one number of each of the 24 different pairs of chromosomes in an N. tabacum monosomic series and also absence of the "S" pair in a nullisome did not affect F-1 protein composition. Absence of the "E" pair caused reduction in the amount of the least acidic of the two kinds of N. tabacum small subunit polypeptides. The question of how many individual progeny of self-fertile hybrids would have to be analyzed to detect segregation of genes coding for F-1 protein small subunit polypeptides, if segregation occurs, was answered by analysis of F1 hybrids between N. otophora and N. tomentosiformis, and two subspecies of N. suaveolens, together with their F2 progeny. In both cases, analysis of 16 progeny was sufficient to demonstrate a segregation pattern of two F1 hybrid type to one each of the two parental types. Therefore, in the absence of segregation, it is likely that coding information for different kinds of F-1 protein small subunit polypeptides is sequestered on heterologous chromosomes, as postulated in previous reports.

  20. Rpa4, a homolog of the 34-kilodalton subunit of the replication protein A complex.

    PubMed Central

    Keshav, K F; Chen, C; Dutta, A

    1995-01-01

    Replication protein A (RPA) is a complex of three polypeptides of 70, 34, and 13 kDa isolated from diverse eukaryotes. The complex is a single-stranded DNA-binding protein essential for simian virus 40-based DNA replication in vitro and for viability in the yeast Saccharomyces cerevisiae. We have identified a new 30-kDa human protein which interacts with the 70- and 13-kDa subunits of RPA, with a yeast two-hybrid/interaction trap method. This protein, Rpa4, has 47% identity with Rpa2, the 34-kDa subunit of RPA. Rpa4 associates with the 70- and 13-kDa subunits to form a trimeric complex capable of binding to single-stranded DNA. Rpa4 is preferentially expressed in placental and colon mucosa tissues. In the placenta, Rpa4 is more abundant than the 70-kDa Rpa1 subunit and is not associated with either Rpa1 or with any other single-stranded DNA-binding protein. In proliferating cells in culture, Rpa4 is considerably less abundant than Rpa1 and Rpa2. Northern (RNA) blot analysis suggest that there are alternatively processed forms of the RPA4 mRNA, and Southern blot analysis indicates that beside RPA4 there may be other members of the RPA2 gene family. PMID:7760808

  1. Regulation of expression of a soybean storage protein subunit gene. Progress report

    SciTech Connect

    Thompson, J.F.; Madison, J.T.

    1984-07-16

    We have found that soybean cotyledons could be cultured in vitro and that the storage proteins were formed essentially as on a plant. When methionine was added to the medium, the cotyledons grew faster, and the methionine content of the protein fraction was increased by over 20 percent. The high methionine content of the protein fraction was found to be due to a shift in the relative amounts of the two major storage proteins. The later effect was the result of methionine treatment suppressing the expression of one storage protein subunit gene. The goal was to determine the mechanism by which methionine is able to regulate the expression of the ..beta..-subunit gene.

  2. Molecular cloning and sequencing of the gene encoding the fimbrial subunit protein of Bacteroides gingivalis.

    PubMed Central

    Dickinson, D P; Kubiniec, M A; Yoshimura, F; Genco, R J

    1988-01-01

    The gene encoding the fimbrial subunit protein of Bacteroides gingivalis 381, fimbrilin, has been cloned and sequenced. The gene was present as a single copy on the bacterial chromosome, and the codon usage in the gene conformed closely to that expected for an abundant protein. The predicted size of the mature protein was 35,924 daltons, and the secretory form may have had a 10-amino-acid, hydrophilic leader sequence similar to the leader sequences of the MePhe fimbriae family. The protein sequence had no marked similarity to known fimbrial sequences, and no homologous sequences could be found in other black-pigmented Bacteroides species, suggesting that fimbrillin represents a class of fimbrial subunit protein of limited distribution. Images PMID:2895100

  3. Calculation of subunit stoichiometry of large, multisubunit proteins from amino acid compositions.

    PubMed

    Kapp, O H; Qabar, A N; Vinogradov, S N

    1990-01-01

    The subunit stoichiometry of a large, multisubunit protein can be determined from the molar amino acid compositions (i amino acids) of the protein and its subunits. The number of copies of the subunits (1, 2, ... j) is calculated by solving all possible combinations of simultaneous equations in j unknowns (i!/j!(i - j)!). Calculations carried out using the published amino acid compositions determined by analysis and the compositions calculated from the sequences for two proteins of known stoichiometry provided the following results: Escherichia coli aspartate transcarbamoylase (R6C6, Mr = 307.5 kDa), R = 5.6 to 6.6 and C = 5.8 to 6.3, and spinach ribulose-bisphosphate carboxylase (L8S8, Mr = 535 kDa), L = 7.3 to 9.1 and S = 5.6 to 10.6. Calculations were also carried out with the amino acid compositions of two much larger proteins, the E. coli pyruvate dehydrogenase complex, Mr = 5280 kDa, subunits E1 (99.5 kDa), E2 (66 kDa), and E3 (50.6 kDa), and the extracellular hemoglobin of Lumbricus terrestris, Mr = 3760 kDa, subunits M (17 kDa), D1 (31 kDa), D2 (37 kDa), and T (51 kDa); the results for PDHase were E1 = 20 to 24, E2 = 18 to 31, E3 = 21 to 33 and those for Lumbricus hemoglobin were M = 34 to 46, D1 = 13 to 19, D2 = 13 to 18, and T = 34 to 36. Although the sample standard deviations of the mean values are generally high, the proposed method works surprisingly well for the two smaller proteins and provides physically reasonable results for the two larger proteins.

  4. Enhancing the thermal stability of avidin. Introduction of disulfide bridges between subunit interfaces.

    PubMed

    Nordlund, Henri R; Laitinen, Olli H; Uotila, Sanna T H; Nyholm, Thomas; Hytönen, Vesa P; Slotte, J Peter; Kulomaa, Markku S

    2003-01-24

    In this study we showed that tetrameric chicken avidin can be stabilized by introducing intermonomeric disulfide bridges between its subunits. These covalent bonds had no major effects on the biotin binding properties of the respective mutants. Moreover, one of the mutants (Avd-ccci) maintained its tetrameric integrity even in denaturing conditions. The new avidin forms Avd-ci and Avd-ccci, which have native --> denatured transition midpoints (T(m)) of 98.6 and 94.7 degrees C, respectively, in the absence of biotin, will find use in applications where extreme stability or minimal leakage of subunits is required. Furthermore, we showed that the intramonomeric disulfide bridges found in the wild-type avidin affect its stability. The mutant Avd-nc, in which this bridge was removed, had a lower T(m) in the absence of biotin than the wild-type avidin but showed comparable stability in the presence of biotin.

  5. Replication protein A subunit 3 and the iron efficiency response in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In soybean [Glycine max (L.) Merr.], iron deficiency results in interveinal chlorosis and decreased photosynthetic capacity, leading to stunting and yield loss. In this study, gene expression analyses investigated the role of soybean replication protein A (RPA) subunits during iron stress. Nine RP...

  6. Insights from the structural analysis of protein heterodimer interfaces.

    PubMed

    Sowmya, Gopichandran; Anita, Sathyanarayanan; Kangueane, Pandjassarame

    2011-05-07

    Protein heterodimer complexes are often involved in catalysis, regulation, assembly, immunity and inhibition. This involves the formation of stable interfaces between the interacting partners. Hence, it is of interest to describe heterodimer interfaces using known structural complexes. We use a non-redundant dataset of 192 heterodimer complex structures from the protein databank (PDB) to identify interface residues and describe their interfaces using amino-acids residue property preference. Analysis of the dataset shows that the heterodimer interfaces are often abundant in polar residues. The analysis also shows the presence of two classes of interfaces in heterodimer complexes. The first class of interfaces (class A) with more polar residues than core but less than surface is known. These interfaces are more hydrophobic than surfaces, where protein-protein binding is largely hydrophobic. The second class of interfaces (class B) with more polar residues than core and surface is shown. These interfaces are more polar than surfaces, where binding is mainly polar. Thus, these findings provide insights to the understanding of protein-protein interactions.

  7. Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans.

    PubMed Central

    Lochrie, M A; Mendel, J E; Sternberg, P W; Simon, M I

    1991-01-01

    A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology. Images PMID:1907494

  8. SMC proteins constitute two subunits of the mammalian recombination complex RC-1.

    PubMed Central

    Jessberger, R; Riwar, B; Baechtold, H; Akhmedov, A T

    1996-01-01

    Recombination protein complex RC-1, purified from calf thymus nuclear extracts, catalyzes cell-free DNA strand transfer and repair of gaps and deletions through DNA recombination. DNA polymerase E, DNA ligase III and a DNA structure-specific endonuclease co-purify with the five polypeptide complex. Here we describe the identification of two hitherto unknown subunits of RC-1. N-terminal amino acid sequences of the 160 and 130 kDa polypeptides display up to 100% identity to proteins of the structural maintenance of chromosomes (SMC) subfamilies 1 and 2. SMC proteins are involved in mitotic chromosome segregation and condensation, as well as in certain DNA repair pathways in fission (rad18 gene) and budding (RHC18 gene) yeast. The assignment was substantiated by immuno-cross-reactivity of the RC-1 subunits with polyclonal antibodies specific for Xenopus laevis SMC proteins. These antibodies, and polyclonal antibodies directed against the bovine 160 and 130 kDa polypeptides, named BSMC1 and BSMC2 (bovine SMC), inhibited RC-1-mediated DNA transfer, indicating that the SMC proteins are necessary components of the reaction. Two independent assays revealed DNA reannealing activity of RC-1, which resides in its BSMC subunits, thereby demonstrating a novel function of these proteins. To our knowledge, this is the first evidence for the association of mammalian SMC proteins with a multiprotein complex harboring, among others, DNA recombination, DNA ligase and DNA polymerase activities. Images PMID:8670910

  9. Structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design.

    PubMed

    Kotecha, Abhay; Seago, Julian; Scott, Katherine; Burman, Alison; Loureiro, Silvia; Ren, Jingshan; Porta, Claudine; Ginn, Helen M; Jackson, Terry; Perez-Martin, Eva; Siebert, C Alistair; Paul, Guntram; Huiskonen, Juha T; Jones, Ian M; Esnouf, Robert M; Fry, Elizabeth E; Maree, Francois F; Charleston, Bryan; Stuart, David I

    2015-10-01

    Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.

  10. Suppression of conformational heterogeneity at a protein-protein interface.

    PubMed

    Deis, Lindsay N; Wu, Qinglin; Wang, You; Qi, Yang; Daniels, Kyle G; Zhou, Pei; Oas, Terrence G

    2015-07-21

    Staphylococcal protein A (SpA) is an important virulence factor from Staphylococcus aureus responsible for the bacterium's evasion of the host immune system. SpA includes five small three-helix-bundle domains that can each bind with high affinity to many host proteins such as antibodies. The interaction between a SpA domain and the Fc fragment of IgG was partially elucidated previously in the crystal structure 1FC2. Although informative, the previous structure was not properly folded and left many substantial questions unanswered, such as a detailed description of the tertiary structure of SpA domains in complex with Fc and the structural changes that take place upon binding. Here we report the 2.3-Å structure of a fully folded SpA domain in complex with Fc. Our structure indicates that there are extensive structural rearrangements necessary for binding Fc, including a general reduction in SpA conformational heterogeneity, freezing out of polyrotameric interfacial residues, and displacement of a SpA side chain by an Fc side chain in a molecular-recognition pocket. Such a loss of conformational heterogeneity upon formation of the protein-protein interface may occur when SpA binds its multiple binding partners. Suppression of conformational heterogeneity may be an important structural paradigm in functionally plastic proteins.

  11. The APC/C subunit Cdc16/Cut9 is a contiguous tetratricopeptide repeat superhelix with a homo-dimer interface similar to Cdc27

    PubMed Central

    Zhang, Ziguo; Kulkarni, Kiran; Hanrahan, Sarah J; Thompson, Andrew J; Barford, David

    2010-01-01

    The anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase responsible for controlling cell cycle transitions, is a multisubunit complex assembled from 13 different proteins. Numerous APC/C subunits incorporate multiple copies of the tetratricopeptide repeat (TPR). Here, we report the crystal structure of Schizosaccharomyces pombe Cut9 (Cdc16/Apc6) in complex with Hcn1 (Cdc26), showing that Cdc16/Cut9 is a contiguous TPR superhelix of 14 TPR units. A C-terminal block of TPR motifs interacts with Hcn1, whereas an N-terminal TPR block mediates Cdc16/Cut9 self-association through a homotypic interface. This dimer interface is structurally related to the N-terminal dimerization domain of Cdc27, demonstrating that both Cdc16/Cut9 and Cdc27 form homo-dimers through a conserved mechanism. The acetylated N-terminal Met residue of Hcn1 is enclosed within a chamber created from the Cut9 TPR superhelix. Thus, in complex with Cdc16/Cut9, the N-acetyl-Met residue of Hcn1, a putative degron for the Doa10 E3 ubiquitin ligase, is inaccessible for Doa10 recognition, protecting Hcn1/Cdc26 from ubiquitin-dependent degradation. This finding may provide a structural explanation for a mechanism to control the stoichiometry of proteins participating in multisubunit complexes. PMID:20924356

  12. Essential functions of the 32 kDa subunit of yeast replication protein A

    PubMed Central

    Dickson, Anne M.; Krasikova, Yulia; Pestryakov, Pavel; Lavrik, Olga; Wold, Marc S.

    2009-01-01

    Replication protein A (RPA) is a heterotrimeric (70, 32 and 14 kDa subunits), single-stranded DNA-binding protein required for cellular DNA metabolism. All subunits of RPA are essential for life, but the specific functions of the 32 and 14 kDa subunits remains unknown. The 32 kDa subunit (RPA2) has multiple domains, but only the central DNA-binding domain (called DBD D) is essential for life in Saccharomyces cerevisiae. To define the essential function(s) of RPA2 in S. cerevisiae, a series of site-directed mutant forms of DBD D were generated. These mutant constructs were then characterized in vitro and in vivo. The mutations had minimal effects on the overall structure and activity of the RPA complex. However, several mutants were shown to disrupt crosslinking of RPA2 to DNA and to dramatically lower the DNA-binding affinity of a RPA2-containing subcomplex. When introduced into S. cerevisiae, all DBD D mutants were viable and supported normal growth rates and DNA replication. These findings indicate that RPA2–DNA interactions are not essential for viability and growth in S. cerevisiae. We conclude that DNA-binding activity of RPA2 is dispensable in yeast and that the essential function of DBD D is intra- and/or inter-protein interactions. PMID:19244309

  13. Regulatory–auxiliary subunits of CLC chloride channel–transport proteins

    PubMed Central

    Barrallo-Gimeno, Alejandro; Gradogna, Antonella; Zanardi, Ilaria; Pusch, Michael; Estévez, Raúl

    2015-01-01

    Abstract The CLC family of chloride channels and transporters is composed by nine members, but only three of them, ClC-Ka/b, ClC-7 and ClC-2, have been found so far associated with auxiliary subunits. These CLC regulatory subunits are small proteins that present few common characteristics among them, both structurally and functionally, and their effects on the corresponding CLC protein are different. Barttin, a protein with two transmembrane domains, is essential for the membrane localization of ClC-K proteins and their activity in the kidney and inner ear. Ostm1 is a protein with a single transmembrane domain and a highly glycosylated N-terminus. Unlike the other two CLC auxiliary subunits, Ostm1 shows a reciprocal relationship with ClC-7 for their stability. The subcellular localization of Ostm1 depends on ClC-7 and not the other way around. ClC-2 is active on its own, but GlialCAM, a transmembrane cell adhesion molecule with two extracellular immunoglobulin (Ig)-like domains, regulates its subcellular localization and activity in glial cells. The common theme for these three proteins is their requirement for a proper homeostasis, since their malfunction leads to distinct diseases. We will review here their properties and their role in normal chloride physiology and the pathological consequences of their improper function. PMID:25762128

  14. Are there proteins between the ribosomal subunits? Hot tritium bombardment experiments.

    PubMed

    Yusupov, M M; Spirin, A S

    1986-03-03

    The hot tritium bombardment technique [(1976) Dokl. Akad. Nauk SSSR 228, 1237-1238] was used for studying the surface localization of ribosomal proteins on Escherichia coli ribosomes. The degree of tritium labeling of proteins was considered as a measure of their exposure (surface localization). Proteins S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27 were shown to be the most exposed on the ribosome surface. The sets of exposed ribosomal proteins on the surface of 70 S ribosomes, on the one hand, and the surfaces of 50 S and 30 S ribosomal subunits in the dissociated state, on the other, were compared. It was found that the dissociation of ribosomes into subunits did not result in exposure of additional ribosomal proteins. The conclusion was drawn that proteins are absent from the contacting surfaces of the ribosomal subunits.

  15. Regulatory-auxiliary subunits of CLC chloride channel-transport proteins.

    PubMed

    Barrallo-Gimeno, Alejandro; Gradogna, Antonella; Zanardi, Ilaria; Pusch, Michael; Estévez, Raúl

    2015-09-15

    The CLC family of chloride channels and transporters is composed by nine members, but only three of them, ClC-Ka/b, ClC-7 and ClC-2, have been found so far associated with auxiliary subunits. These CLC regulatory subunits are small proteins that present few common characteristics among them, both structurally and functionally, and their effects on the corresponding CLC protein are different. Barttin, a protein with two transmembrane domains, is essential for the membrane localization of ClC-K proteins and their activity in the kidney and inner ear. Ostm1 is a protein with a single transmembrane domain and a highly glycosylated N-terminus. Unlike the other two CLC auxiliary subunits, Ostm1 shows a reciprocal relationship with ClC-7 for their stability. The subcellular localization of Ostm1 depends on ClC-7 and not the other way around. ClC-2 is active on its own, but GlialCAM, a transmembrane cell adhesion molecule with two extracellular immunoglobulin (Ig)-like domains, regulates its subcellular localization and activity in glial cells. The common theme for these three proteins is their requirement for a proper homeostasis, since their malfunction leads to distinct diseases. We will review here their properties and their role in normal chloride physiology and the pathological consequences of their improper function.

  16. The Alpha Catalytic Subunit of Protein Kinase CK2 Is Required for Mouse Embryonic Development▿

    PubMed Central

    Lou, David Y.; Dominguez, Isabel; Toselli, Paul; Landesman-Bollag, Esther; O'Brien, Conor; Seldin, David C.

    2008-01-01

    Protein kinase CK2 (formerly casein kinase II) is a highly conserved and ubiquitous serine/threonine kinase that is composed of two catalytic subunits (CK2α and/or CK2α′) and two CK2β regulatory subunits. CK2 has many substrates in cells, and key roles in yeast cell physiology have been uncovered by introducing subunit mutations. Gene-targeting experiments have demonstrated that in mice, the CK2β gene is required for early embryonic development, while the CK2α′ subunit appears to be essential only for normal spermatogenesis. We have used homologous recombination to disrupt the CK2α gene in the mouse germ line. Embryos lacking CK2α have a marked reduction in CK2 activity in spite of the presence of the CK2α′ subunit. CK2α−/− embryos die in mid-gestation, with abnormalities including open neural tubes and reductions in the branchial arches. Defects in the formation of the heart lead to hydrops fetalis and are likely the cause of embryonic lethality. Thus, CK2α appears to play an essential and uncompensated role in mammalian development. PMID:17954558

  17. Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing

    SciTech Connect

    Kvissel, Anne-Katrine . E-mail: a.k.kvissel@basalmed.uio.no; Orstavik, Sigurd; Eikvar, Sissel; Brede, Gaute; Jahnsen, Tore; Collas, Philippe; Akusjaervi, Goeran; Skalhegg, Bjorn Steen

    2007-08-01

    Protein kinase A (PKA) is a holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer. Stimulation by cAMP dissociates the holoenzyme and causes translocation to the nucleus of a fraction of the C subunit. Apart from transcription regulation, little is known about the function of the C subunit in the nucleus. In the present report, we show that both C{alpha} and C{beta} are localized to spots in the mammalian nucleus. Double immunofluorescence analysis of splicing factor SC35 with the C subunit indicated that these spots are splicing factor compartments (SFCs). Using the E1A in vivo splicing assay, we found that catalytically active C subunits regulate alternative splicing and phosphorylate several members of the SR-protein family of splicing factors in vitro. Furthermore, nuclear C subunits co-localize with the C subunit-binding protein homologous to AKAP95, HA95. HA95 also regulates E1A alternative splicing in vivo, apparently through its N-terminal domain. Localization of the C subunit to SFCs and the E1A splicing pattern were unaffected by cAMP stimulation. Our findings demonstrate that the nuclear PKA C subunit co-locates with HA95 in SFCs and regulates pre-mRNA splicing, possibly through a cAMP-independent mechanism.

  18. Interactions of protein content and globulin subunit composition of soybean proteins in relation to tofu gel properties.

    PubMed

    James, Andrew T; Yang, Aijun

    2016-03-01

    The content and globulin subunit composition of soybean proteins are known to affect tofu quality and food-grade soybeans usually have higher levels of proteins. We studied the tofu quality of soybeans with high (44.8%) or low (39.1%) protein content and with or without the 11S globulin polypeptide, 11SA4. Both protein content and 11SA4 significantly affected tofu gel properties. Soybeans containing more protein had smaller seeds which produced significantly firmer (0.663 vs.0.557 N, p<0.001) tofu gels with creamier colour. The absence of 11SA4 was positively correlated with seed size, tofu hardness and water holding capacity and led to significant changes to the profile of storage protein subunits, which may have contributed to the improvement in tofu gel properties. These results suggest that, in combination with higher protein content, certain protein subunits or their polypeptides can also be targeted in selecting soybeans to further improve soy food quality.

  19. Proliferation of transformed somatotroph cells related to low or absent expression of protein kinase a regulatory subunit 1A protein.

    PubMed

    Lania, Andrea G; Mantovani, Giovanna; Ferrero, Stefano; Pellegrini, Caterina; Bondioni, Sara; Peverelli, Erika; Braidotti, Paola; Locatelli, Marco; Zavanone, Mario L; Ferrante, Emanuela; Bosari, Silvano; Beck-Peccoz, Paolo; Spada, Anna

    2004-12-15

    The two regulatory subunits (R1 and R2) of protein kinase A (PKA) are differentially expressed in cancer cell lines and exert diverse roles in growth control. Recently, mutations of the PKA regulatory subunit 1A gene (PRKAR1A) have been identified in patients with Carney complex. The aim of this study was to evaluate the expression of the PKA regulatory subunits R1A, R2A, and R2B in a series of 30 pituitary adenomas and the effects of subunit activation on cell proliferation. In these tumors, neither mutation of PRKAR1A nor loss of heterozygosity was identified. By real-time PCR, mRNA of the three subunits was detected in all of the tumors, R1A being the most represented in the majority of samples. By contrast, immunohistochemistry documented low or absent R1A levels in all tumors, whereas R2A and R2B were highly expressed, thus resulting in an unbalanced R1/R2 ratio. The low levels of R1A were, at least in part, due to proteasome-mediated degradation. The effect of the R1/R2 ratio on proliferation was assessed in GH3 cells, which showed a similar unbalanced pattern of R subunits expression, and in growth hormone-secreting adenomas. The R2-selective cAMP analog 8-Cl cAMP and R1A RNA silencing, stimulated cell proliferation and increased Cyclin D1 expression, respectively, in human and rat adenomatous somatotrophs. These data show that a low R1/R2 ratio promoted proliferation of transformed somatotrophs and are consistent with the Carney complex model in which R1A inactivating mutations further unbalance this ratio in favor of R2 subunits. These results suggest that low expression of R1A protein may favor cAMP-dependent proliferation of transformed somatotrophs.

  20. Adjuvant effect of the human metapneumovirus (HMPV) matrix protein in HMPV subunit vaccines.

    PubMed

    Aerts, Laetitia; Rhéaume, Chantal; Carbonneau, Julie; Lavigne, Sophie; Couture, Christian; Hamelin, Marie-Ève; Boivin, Guy

    2015-04-01

    The human metapneumovirus (HMPV) fusion (F) protein is the most immunodominant protein, yet subunit vaccines containing only this protein do not confer complete protection. The HMPV matrix (M) protein induces the maturation of antigen-presenting cells in vitro. The inclusion of the M protein into an F protein subunit vaccine might therefore provide an adjuvant effect. We administered the F protein twice intramuscularly, adjuvanted with alum, the M protein or both, to BALB/c mice at 3 week intervals. Three weeks after the boost, mice were infected with HMPV and monitored for 14 days. At day 5 post-challenge, pulmonary viral titres, histopathology and cytokine levels were analysed. Mice immunized with F+alum and F+M+alum generated significantly more neutralizing antibodies than mice immunized with F only [titres of 47 ± 7 (P<0.01) and 147 ± 13 (P<0.001) versus 17 ± 2]. Unlike F only [1.6 ± 0.5 × 10(3) TCID50 (g lung)(-1)], pulmonary viral titres in mice immunized with F+M and F+M+alum were undetectable. Mice immunized with F+M presented the most important reduction in pulmonary inflammation and the lowest T-helper Th2/Th1 cytokine ratio. In conclusion, addition of the HMPV-M protein to an F protein-based vaccine modulated both humoral and cellular immune responses to subsequent infection, thereby increasing the protection conferred by the vaccine.

  1. Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats

    NASA Technical Reports Server (NTRS)

    Mednieks, M. I.; Popova, I.; Grindeland, R. E.

    1992-01-01

    Photoaffinity labeling in heart tissue of rats flown on Cosmos 2044 was used to measure the regulatory (R) subunits of adenosine monophosphate-dependent protein kinase. A significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) was observed when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was observed to be unaffected by spaceflight or any of the simulation conditions. Proteins of the S2 fraction constitute a minor (less than 10 percent) component of the total, whereas the S1 fraction contained most of the cell proteins. Changes in a relatively minor aspect of adenosine monophosphate-mediated reactions are considered to be representative of a metabolic effect.

  2. Assembly of transmembrane proteins on oil-water interfaces

    NASA Astrophysics Data System (ADS)

    Yunker, Peter; Landry, Corey; Chong, Shaorong; Weitz, David

    2015-03-01

    Transmembrane proteins are difficult to handle by aqueous solution-based biochemical and biophysical approaches, due to the hydrophobicity of transmembrane helices. Detergents can solubilize transmembrane proteins; however, surfactant coated transmembrane proteins are not always functional, and purifying detergent coated proteins in a micellar solution can be difficult. Motivated by this problem, we study the self-assembly of transmembrane proteins on oil-water interfaces. We found that the large water-oil interface of oil drops prevents nascent transmembrane proteins from forming non-functional aggregates. The oil provides a hydrophobic environment for the transmembrane helix, allowing the ectodomain to fold into its natural structure and orientation. Further, modifying the strength or valency of hydrophobic interactions between transmembrane proteins results in the self-assembly of spatially clustered, active proteins on the oil-water interface. Thus, hydrophobic interactions can facilitate, rather than inhibit, the assembly of transmembrane proteins.

  3. Molecular chaperoning function of Ric-8 is to fold nascent heterotrimeric G protein α subunits.

    PubMed

    Chan, Puiyee; Thomas, Celestine J; Sprang, Stephen R; Tall, Gregory G

    2013-03-05

    We have shown that resistance to inhibitors of cholinesterase 8 (Ric-8) proteins regulate an early step of heterotrimeric G protein α (Gα) subunit biosynthesis. Here, mammalian and plant cell-free translation systems were used to study Ric-8A action during Gα subunit translation and protein folding. Gα translation rates and overall produced protein amounts were equivalent in mock and Ric-8A-immunodepleted rabbit reticulocyte lysate (RRL). GDP-AlF4(-)-bound Gαi, Gαq, Gα13, and Gαs produced in mock-depleted RRL had characteristic resistance to limited trypsinolysis, showing that these G proteins were folded properly. Gαi, Gαq, and Gα13, but not Gαs produced from Ric-8A-depleted RRL were not protected from trypsinization and therefore not folded correctly. Addition of recombinant Ric-8A to the Ric-8A-depleted RRL enhanced GDP-AlF4(-)-bound Gα subunit trypsin protection. Dramatic results were obtained in wheat germ extract (WGE) that has no endogenous Ric-8 component. WGE-translated Gαq was gel filtered and found to be an aggregate. Ric-8A supplementation of WGE allowed production of Gαq that gel filtered as a ∼100 kDa Ric-8A:Gαq heterodimer. Addition of GTPγS to Ric-8A-supplemented WGE Gαq translation resulted in dissociation of the Ric-8A:Gαq heterodimer and production of functional Gαq-GTPγS monomer. Excess Gβγ supplementation of WGE did not support functional Gαq production. The molecular chaperoning function of Ric-8 is to participate in the folding of nascent G protein α subunits.

  4. A 102 kDa subunit of a Golgi-associated particle has homology to beta subunits of trimeric G proteins.

    PubMed Central

    Harrison-Lavoie, K J; Lewis, V A; Hynes, G M; Collison, K S; Nutland, E; Willison, K R

    1993-01-01

    We have identified a 102 kDa protein, p102, which is found on the cytoplasmic face of Golgi membranes, exocytic transport vesicles and in the cytosol. A monoclonal antibody that cross-reacts with p102 is able to immunoprecipitate a 500-600 kDa protein complex containing p102 and additional subunits. The composition of this p102-containing protein complex resembles that of the Golgi coatomer complex, which constitutes the coat of non-clathrin coated vesicles. One of the subunits of the p102 complex reacts with a monoclonal antibody that detects beta-COP, a subunit of the Golgi coatomer complex. Like beta-COP, p102 exists in a brefeldin A-sensitive association with Golgi membranes. The sequence of p102 contains an N-terminal domain composed of six repeats which are similar to those found in the beta subunit of trimeric G proteins and other regulatory proteins. We suggest that p102 may be involved in regulating membrane traffic in the constitutive exocytic pathway. Images PMID:8335000

  5. Distribution of regulatory subunits of protein kinase A and A kinase anchor proteins (AKAP 95, 150) in rat pinealocytes.

    PubMed

    Koch, M; Korf, H-W

    2002-12-01

    The rat pineal organ is an established model to study signal transduction cascades that are activated by norepinephrine (NE) and cause increases in cAMP levels and stimulation of protein kinase A (PKA). PKA type II catalyzes the phosphorylation of the transcription factor cAMP-response-element-binding protein (CREB) which is essential for the transcriptional induction of the arylalkylamine- N-acetyltransferase (AANAT), the rate limiting enzyme of melatonin biosynthesis. Moreover, PKA may control protein levels and enzyme activity via two PKA-dependent phosphorylation sites in the AANAT molecule. Despite the functional importance of PKA very little is known about the distribution of its isoenzymes and of A-kinase anchor proteins (AKAPs) that target the PKA to specific membrane areas and organelles by binding to the regulatory (R) subunits of PKA. We have addressed this problem by demonstrating the R subunits alpha and beta of PKA type I and II and two AKAPs (150 and 95) in NE-stimulated and untreated rat pinealocytes by immunoblot and immunocytochemistry. The immunoreactions (IR) of all four R subunits were confined to granules evenly distributed in the pinealocyte cytoplasm. Immunoreactions of RIIalpha and RIIbeta were stronger than those of RIalpha and RIbeta. AKAP 150-IR was concentrated at the cell periphery; AKAP 95-IR was restricted to the nucleus. Amount and subcellular distribution of the immunoreactions of all proteins investigated did not change upon NE stimulation. A substantial colocalization was observed between RII-subunits and AKAP 150-IR, suggesting that, in rat pinealocytes, AKAP 150 primarily anchors the R subunits of PKA II.

  6. G alpha 16, a G protein alpha subunit specifically expressed in hematopoietic cells.

    PubMed Central

    Amatruda, T T; Steele, D A; Slepak, V Z; Simon, M I

    1991-01-01

    Signal-transduction pathways mediated by guanine nucleotide-binding regulatory proteins (G proteins) determine many of the responses of hematopoietic cells. A recently identified gene encoding a G protein alpha subunit, G alpha 16, is specifically expressed in human cells of the hematopoietic lineage. The G alpha 16 cDNA encodes a protein with predicted Mr of 43,500, which resembles the G q class of alpha subunits and does not include a pertussis toxin ADP-ribosylation site. In comparison with other G protein alpha subunits, the G alpha 16 predicted protein has distinctive amino acid sequences in the amino terminus, the region A guanine nucleotide-binding domain, and in the carboxyl-terminal third of the protein. Cell lines of myelomonocytic and T-cell phenotype express the G alpha 16 gene, but no expression is detectable in two B-cell lines or in nonhematopoietic cell lines. G alpha 16 gene expression is down-regulated in HL-60 cells induced to differentiate to neutrophils with dimethyl sulfoxide. Antisera generated from synthetic peptides that correspond to two regions of G alpha 16 specifically react with a protein of 42- to 43-kDa in bacterial strains that overexpress G alpha 16 and in HL-60 membranes. This protein is decreased in membranes from dimethyl sulfoxide-differentiated HL-60 cells and is not detectable in COS cell membranes. The restricted expression of this gene suggests that G alpha 16 regulates cell-type-specific signal-transduction pathways, which are not inhibited by pertussis toxin. Images PMID:1905813

  7. Structure-function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring.

    PubMed

    Schwer, Beate; Kruchten, Joshua; Shuman, Stewart

    2016-09-01

    A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure-function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein-RNA and protein-protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not.

  8. Shared Protein Complex Subunits Contribute to Explaining Disrupted Co-occurrence

    PubMed Central

    Schneider, Adrian; Seidl, Michael F.; Snel, Berend

    2013-01-01

    The gene composition of present-day genomes has been shaped by a complicated evolutionary history, resulting in diverse distributions of genes across genomes. The pattern of presence and absence of a gene in different genomes is called its phylogenetic profile. It has been shown that proteins whose encoding genes have highly similar profiles tend to be functionally related: As these genes were gained and lost together, their encoded proteins can probably only perform their full function if both are present. However, a large proportion of genes encoding interacting proteins do not have matching profiles. In this study, we analysed one possible reason for this, namely that phylogenetic profiles can be affected by multi-functional proteins such as shared subunits of two or more protein complexes. We found that by considering triplets of proteins, of which one protein is multi-functional, a large fraction of disturbed co-occurrence patterns can be explained. PMID:23874172

  9. Calmodulin-dependent protein phosphatase from Neurospora crassa. Molecular cloning and expression of recombinant catalytic subunit.

    PubMed

    Higuchi, S; Tamura, J; Giri, P R; Polli, J W; Kincaid, R L

    1991-09-25

    A cDNA for the catalytic subunit of a calmodulin (CaM)-dependent protein phosphatase was cloned from Neurospora crassa. The open reading frame of 1557 base pairs encoded a protein of Mr approximately 59,580 and was followed by a 3'-untranslated region of 363 base pairs including the poly(A) tail. Based on primer extension analysis, the mRNA transcript in vivo was 2403 base pairs. Expression of this CaM-protein phosphatase mRNA was developmentally regulated, being highest during early mycelial growth; production of the corresponding protein followed mRNA with a time lag of 8-12 h. Polymerase chain reaction amplification of genomic DNA revealed three small introns, the positions of which coincided with those in the mouse gene, indicating evolutionary conservation of these structures. The deduced sequence showed approximately 75% identity with the mammalian homologue, calcineurin, in aligned regions. A region of 40 amino acids preceding the CaM-binding domain was essentially unchanged, suggesting conservation of a crucial interaction site. Three small segments in the carboxyl half of the protein were unrelated to the mammalian gene and may constitute "variable regions" that confer substrate specificity to the enzyme. An active recombinant catalytic subunit was expressed in bacteria and purified by CaM-Sepharose chromatography. This preparation was stimulated 2- 3-fold by CaM and showed a p-nitrophenol phosphatase activity equal to that of the bovine brain holoenzyme, although its dephosphorylation of phosphoprotein substrates was markedly different. These findings demonstrate that the catalytic subunit of this phosphatase can exhibit high activity in the absence of its intrinsic Ca(2+)-binding subunit.

  10. NSs protein of rift valley fever virus promotes posttranslational downregulation of the TFIIH subunit p62.

    PubMed

    Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

    2011-07-01

    Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus.

  11. A G-Protein Subunit Translocation Embedded Network Motif Underlies GPCR Regulation of Calcium Oscillations

    PubMed Central

    Giri, Lopamudra; Patel, Anilkumar K.; Karunarathne, W.K. Ajith; Kalyanaraman, Vani; Venkatesh, K.V.; Gautam, N.

    2014-01-01

    G-protein βγ subunits translocate reversibly from the plasma membrane to internal membranes on receptor activation. Translocation rates differ depending on the γ subunit type. There is limited understanding of the role of the differential rates of Gβγ translocation in modulating signaling dynamics in a cell. Bifurcation analysis of the calcium oscillatory network structure predicts that the translocation rate of a signaling protein can regulate the damping of system oscillation. Here, we examined whether the Gβγ translocation rate regulates calcium oscillations induced by G-protein-coupled receptor activation. Oscillations in HeLa cells expressing γ subunit types with different translocation rates were imaged and quantitated. The results show that differential Gβγ translocation rates can underlie the diversity in damping characteristics of calcium oscillations among cells. Mathematical modeling shows that a translocation embedded motif regulates damping of G-protein-mediated calcium oscillations consistent with experimental data. The current study indicates that such a motif may act as a tuning mechanism to design oscillations with varying damping patterns by using intracellular translocation of a signaling component. PMID:24988358

  12. Evolutionary and structural analyses of heterodimeric proteins composed of subunits with same fold.

    PubMed

    Sudha, Govindarajan; Naveenkumar, Nagarajan; Srinivasan, Narayanaswamy

    2015-10-01

    Heterodimeric proteins with homologous subunits of same fold are involved in various biological processes. The objective of this study is to understand the evolution of structural and functional features of such heterodimers. Using a non-redundant dataset of 70 such heterodimers of known 3D structure and an independent dataset of 173 heterodimers from yeast, we note that the mean sequence identity between interacting homologous subunits is only 23-24% suggesting that, generally, highly diverged paralogues assemble to form such a heterodimer. We also note that the functional roles of interacting subunits/domains are generally quite different. This suggests that, though the interacting subunits/domains are homologous, the high evolutionary divergence characterize their high functional divergence which contributes to a gross function for the heterodimer considered as a whole. The inverse relationship between sequence identity and RMSD of interacting homologues in heterodimers is not followed. We also addressed the question of formation of homodimers of the subunits of heterodimers by generating models of fictitious homodimers on the basis of the 3D structures of the heterodimers. Interaction energies associated with these homodimers suggests that, in overwhelming majority of the cases, such homodimers are unlikely to be stable. Majority of the homologues of heterodimers of known structures form heterodimers (51.8%) and a small proportion (14.6%) form homodimers. Comparison of 3D structures of heterodimers with homologous homodimers suggests that interfacial nature of residues is not well conserved. In over 90% of the cases we note that the interacting subunits of heterodimers are co-localized in the cell.

  13. Studies on the Assembly Characteristics of Large Subunit Ribosomal Proteins in S. cerevisae

    PubMed Central

    Ohmayer, Uli; Gamalinda, Michael; Sauert, Martina; Ossowski, Julius; Pöll, Gisela; Linnemann, Jan; Hierlmeier, Thomas; Perez-Fernandez, Jorge; Kumcuoglu, Beril; Leger-Silvestre, Isabelle; Faubladier, Marlène; Griesenbeck, Joachim; Woolford, John; Tschochner, Herbert; Milkereit, Philipp

    2013-01-01

    During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed. PMID:23874617

  14. Protein-Protein Interface Detection Using the Energy Centrality Relationship (ECR) Characteristic of Proteins

    PubMed Central

    Sudarshan, Sanjana; Kodathala, Sasi B.; Mahadik, Amruta C.; Mehta, Isha; Beck, Brian W.

    2014-01-01

    Specific protein interactions are responsible for most biological functions. Distinguishing Functionally Linked Interfaces of Proteins (FLIPs), from Functionally uncorrelated Contacts (FunCs), is therefore important to characterizing these interactions. To achieve this goal, we have created a database of protein structures called FLIPdb, containing proteins belonging to various functional sub-categories. Here, we use geometric features coupled with Kortemme and Baker's computational alanine scanning method to calculate the energetic sensitivity of each amino acid at the interface to substitution, identify hotspots, and identify other factors that may contribute towards an interface being FLIP or FunC. Using Principal Component Analysis and K-means clustering on a training set of 160 interfaces, we could distinguish FLIPs from FunCs with an accuracy of 76%. When these methods were applied to two test sets of 18 and 170 interfaces, we achieved similar accuracies of 78% and 80%. We have identified that FLIP interfaces have a stronger central organizing tendency than FunCs, due, we suggest, to greater specificity. We also observe that certain functional sub-categories, such as enzymes, antibody-heavy-light, antibody-antigen, and enzyme-inhibitors form distinct sub-clusters. The antibody-antigen and enzyme-inhibitors interfaces have patterns of physical characteristics similar to those of FunCs, which is in agreement with the fact that the selection pressures of these interfaces is differently evolutionarily driven. As such, our ECR model also successfully describes the impact of evolution and natural selection on protein-protein interfaces. Finally, we indicate how our ECR method may be of use in reducing the false positive rate of docking calculations. PMID:24830938

  15. Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein-protein interfaces.

    PubMed

    Wylie, Benjamin J; Dzikovski, Boris G; Pawsey, Shane; Caporini, Marc; Rosay, Melanie; Freed, Jack H; McDermott, Ann E

    2015-04-01

    We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces.

  16. Rnr4p, a novel ribonucleotide reductase small-subunit protein.

    PubMed Central

    Wang, P J; Chabes, A; Casagrande, R; Tian, X C; Thelander, L; Huffaker, T C

    1997-01-01

    Ribonucleotide reductases catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. Eukaryotic ribonucleotide reductases are alpha2beta2 tetramers; each of the larger, alpha subunits possesses binding sites for substrate and allosteric effectors, and each of the smaller, beta subunits contains a binuclear iron complex. The iron complex interacts with a specific tyrosine residue to form a tyrosyl free radical which is essential for activity. Previous work has identified two genes in the yeast Saccharomyces cerevisiae, RNR1 and RNR3, that encode alpha subunits and one gene, RNR2, that encodes a beta subunit. Here we report the identification of a second gene from this yeast, RNR4, that encodes a protein with significant similarity to the beta-subunit proteins. The phenotype of rnr4 mutants is consistent with that expected for a defect in ribonucleotide reductase; rnr4 mutants are supersensitive to the ribonucleotide reductase inhibitor hydroxyurea and display an S-phase arrest at their restrictive temperature. rnr4 mutant extracts are deficient in ribonucleotide reductase activity, and this deficiency can be remedied by the addition of exogenous Rnr4p. As is the case for the other RNR genes, RNR4 is induced by agents that damage DNA. However, Rnr4p lacks a number of sequence elements thought to be essential for iron binding, and mutation of the critical tyrosine residue does not affect Rnr4p function. These results suggest that Rnr4p is catalytically inactive but, nonetheless, does play a role in the ribonucleotide reductase complex. PMID:9315671

  17. The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

    NASA Technical Reports Server (NTRS)

    Deruere, J.; Jackson, K.; Garbers, C.; Soll, D.; Delong, A.; Evans, M. L. (Principal Investigator)

    1999-01-01

    Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

  18. The CDC2-related kinase PITALRE is the catalytic subunit of active multimeric protein complexes.

    PubMed Central

    Garriga, J; Mayol, X; Graña, X

    1996-01-01

    PITALRE is a human protein kinase identified by means of its partial sequence identity to the cell division cycle regulatory kinase CDC2. Immunopurified PITALRE protein complexes exhibit an in vitro kinase activity that phosphorylates the retinoblastoma protein, suggesting that PITALRE catalyses this phosphorylation reaction. However, the presence of other kinases in the immunopurified complex could not be ruled out. In the present work, an inactive mutant of the PITALRE kinase has been used to demonstrate that PITALRE is the catalytic subunit responsible for the PITALRE-complex-associated kinase activity, Ectopic overexpression of PITALRE did not increase the total PITALRE kinase activity in the cell, suggesting that PITALRE is regulated by limiting cellular factor(s). Characterization of the PITALRE-containing protein complexes indicated that most of the cellular PITALRE protein exists as a subunit in at least two different active multimeric complexes. Although monomeric PITALRE is also active in vitro, PITALRE present in multimeric complexes exhibits several-fold higher activity than monomeric PITALRE. In addition, overexpression of PITALRE demonstrated the existence of two new associated proteins of approx. 48 and 98 kDa. Altogether these results suggest that, in contrast to the situation with cyclin-dependent kinases, monomeric PITALRE is active, and that association with other proteins modulates its activity and/or its ability to recognize substrates in vivo. PMID:8870681

  19. PI2PE: protein interface/interior prediction engine.

    PubMed

    Tjong, Harianto; Qin, Sanbo; Zhou, Huan-Xiang

    2007-07-01

    The side chains of the 20 types of amino acids, owing to a large extent to their different physical properties, have characteristic distributions in interior/surface regions of individual proteins and in interface/non-interface portions of protein surfaces that bind proteins or nucleic acids. These distributions have important structural and functional implications. We have developed accurate methods for predicting the solvent accessibility of amino acids from a protein sequence and for predicting interface residues from the structure of a protein-binding or DNA-binding protein. The methods are called WESA, cons-PPISP and DISPLAR, respectively. The web servers of these methods are now available at http://pipe.scs.fsu.edu. To illustrate the utility of these web servers, cons-PPISP and DISPLAR predictions are used to construct a structural model for a multicomponent protein-DNA complex.

  20. Yeast ribosomal protein L10 helps coordinate tRNA movement through the large subunit

    PubMed Central

    Petrov, Alexey N.; Meskauskas, Arturas; Roshwalb, Sara C.; Dinman, Jonathan D.

    2008-01-01

    Yeast ribosomal protein L10 (E. coli L16) is located at the center of a topological nexus that connects many functional regions of the large subunit. This essential protein has previously been implicated in processes as diverse as ribosome biogenesis, translational fidelity and mRNA stability. Here, the inability to maintain the yeast Killer virus was used as a proxy for large subunit defects to identify a series of L10 mutants. These mapped to roughly four discrete regions of the protein. A detailed analysis of mutants located in the N-terminal ‘hook’ of L10, which inserts into the bulge of 25S rRNA helix 89, revealed strong effects on rRNA structure corresponding to the entire path taken by the tRNA 3′ end as it moves through the large subunit during the elongation cycle. The mutant-induced structural changes are wide-ranging, affecting ribosome biogenesis, elongation factor binding, drug resistance/hypersensitivity, translational fidelity and virus maintenance. The importance of L10 as a potential transducer of information through the ribosome, and of a possible role of its N-terminal domain in switching between the pre- and post-translocational states are discussed. PMID:18824477

  1. Myristoylation of an inhibitory GTP-binding protein. alpha. subunit is essential for its membrane attachment

    SciTech Connect

    Jones, T.L.Z.; Simonds, W.F.; Merendino, J.J. Jr.; Brann, M.R.; Spiegel, A.M. )

    1990-01-01

    The authors transfected COS cells with cDNAs for the {alpha} subunits of stimulatory and inhibitory GTP-binding proteins, {alpha}{sub s} and {alpha}{sub i1}, respectively, and immunoprecipitated the metabolically labeled products with specific peptide antibodies. Cells were separated into particulate and soluble fractions before immunoprecipitation; ({sup 35}S)methionine-labeled {alpha}{sub s} and {alpha}{sub i} were both found primarily in the particulate fraction. ({sup 3}H)Myristate was incorporated into endogenous and transfected {alpha}{sub i} but could not be detected in {alpha}{sub s} even when it was overexpressed. They converted the second residue, glycine, of {alpha}{sub i1} into alanine by site-directed mutagenesis. Upon transfection of the mutant {alpha}{sub i1} into COS cells, the ({sup 35}S)methionine-labeled product was localized primarily to the soluble fraction, and, also unlike normal {alpha}{sub i1}, the mutant failed to incorporate ({sup 3}H)myristate. The unmyristoylated mutant {alpha}{sub i1} could still interact with the {beta}-{gamma} complex, since purified {beta}{gamma} subunits promoted pertussis toxin-catalyzed ADP-ribosylation of both the normal and mutant {alpha}{sub i1} subunits. These results indicate that myristoylation is critical for membrane attachment of {alpha}{sub i} but not {alpha}{sub s} subunits.

  2. Enzymatic assembly of epothilones: the EpoC subunit and reconstitution of the EpoA-ACP/B/C polyketide and nonribosomal peptide interfaces.

    PubMed

    O'Connor, Sarah E; Chen, Huawei; Walsh, Christopher T

    2002-04-30

    The biosynthesis of epothilones, a family of hybrid polyketide (PK)/nonribosomal peptide (NRP) antitumor agents, provides an ideal system to study a hybrid PK/NRP natural product with significant biomedical value. Here the third enzyme involved in epothilone production, the five domain 195 kDa polyketide synthase (PKS) EpoC protein, has been expressed and purified from Escherichia coli. EpoC was combined with the first two enzymes of the epothilone biosynthesis pathway, the acyl carrier protein (ACP) domain of EpoA and EpoB, to reconstitute the early steps in epothilone biosynthesis. The acyltransferase (AT) domain of EpoC transfers the methylmalonyl moiety from methylmalonyl-CoA to the holo HS-acyl carrier protein (ACP) in an autoacylation reaction. The ketosynthase (KS) domain of EpoC decarboxylates the methylmalonyl-S-EpoC acyl enzyme to generate the carbon nucleophile that reacts with methylthiazolylcarboxyl-S-EpoB. The resulting condensation product can be reduced in the presence of NADPH by the ketoreductase (KR) domain of EpoC and then dehydrated by the dehydratase (DH) domain to produce the methylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediate that serves as the acyl donor for subsequent elongation of the epothilone chain. The acetyl-CoA donor can be replaced with propionyl-CoA, isobutyryl-CoA, and benzoyl-CoA and the acyl chains accepted by both EpoB and EpoC subunits to produce ethyl-, isopropyl-, and phenylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediates, suggesting that future combinatorial biosynthetic variations in epothilone assembly may be feasible. These results demonstrate in vitro reconstitution of both the PKS/NRPS interface (EpoA-ACP/B) and the NRPS/PKS interface (EpoB/C) in the assembly line for this antitumor natural product.

  3. Size-dependent protein segregation at membrane interfaces

    NASA Astrophysics Data System (ADS)

    Schmid, Eva M.; Bakalar, Matthew H.; Choudhuri, Kaushik; Weichsel, Julian; Ann, Hyoung Sook; Geissler, Phillip L.; Dustin, Michael L.; Fletcher, Daniel A.

    2016-07-01

    Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane proteins whose organization is critical for intracellular signalling. To isolate the role of membrane protein size in pattern formation, we reconstituted model membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between membrane proteins can drastically alter their organization at membrane interfaces, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally driven membrane height fluctuations that transiently limit access to the interface. This sensitive and highly effective means of physically segregating proteins has implications for cell-cell contacts such as T-cell immunological synapses (for example, CD45 exclusion) and epithelial cell junctions (for example, E-cadherin enrichment), as well as for protein sorting at intracellular contact points between membrane-bound organelles.

  4. Biotechnology approaches to produce potent, self-adjuvanting antigen-adjuvant fusion protein subunit vaccines.

    PubMed

    Moyle, Peter Michael

    Traditional vaccination approaches (e.g. live attenuated or killed microorganisms) are among the most effective means to prevent the spread of infectious diseases. These approaches, nevertheless, have failed to yield successful vaccines against many important pathogens. To overcome this problem, methods have been developed to identify microbial components, against which protective immune responses can be elicited. Subunit antigens identified by these approaches enable the production of defined vaccines, with improved safety profiles. However, they are generally poorly immunogenic, necessitating their administration with potent immunostimulatory adjuvants. Since few safe and effective adjuvants are currently used in vaccines approved for human use, with those available displaying poor potency, or an inability to stimulate the types of immune responses required for vaccines against specific diseases (e.g. cytotoxic lymphocytes (CTLs) to treat cancers), the development of new vaccines will be aided by the availability of characterized platforms of new adjuvants, improving our capacity to rationally select adjuvants for different applications. One such approach, involves the addition of microbial components (pathogen-associated molecular patterns; PAMPs), that can stimulate strong immune responses, into subunit vaccine formulations. The conjugation of PAMPs to subunit antigens provides a means to greatly increase vaccine potency, by targeting immunostimulation and antigen to the same antigen presenting cell. Thus, methods that enable the efficient, and inexpensive production of antigen-adjuvant fusions represent an exciting mean to improve immunity towards subunit antigens. Herein we review four protein-based adjuvants (flagellin, bacterial lipoproteins, the extra domain A of fibronectin (EDA), and heat shock proteins (Hsps)), which can be genetically fused to antigens to enable recombinant production of antigen-adjuvant fusion proteins, with a focus on their

  5. Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly.

    PubMed

    Mueller, Susanne; Wahlander, Asa; Selevsek, Nathalie; Otto, Claudia; Ngwa, Elsy Mankah; Poljak, Kristina; Frey, Alexander D; Aebi, Markus; Gauss, Robert

    2015-07-15

    Protein degradation is essential for cellular homeostasis. We developed a sensitive approach to examining protein degradation rates in Saccharomyces cerevisiae by coupling a SILAC approach to selected reaction monitoring (SRM) mass spectrometry. Combined with genetic tools, this analysis made it possible to study the assembly of the oligosaccharyl transferase complex. The ER-associated degradation machinery compensated for disturbed homeostasis of complex components by degradation of subunits in excess. On a larger scale, protein degradation in the ER was found to be a minor factor in the regulation of protein homeostasis in exponentially growing cells, but ERAD became relevant when the gene dosage was affected, as demonstrated in heterozygous diploid cells. Hence the alleviation of fitness defects due to abnormal gene copy numbers might be an important function of protein degradation.

  6. Changes in protein structure at the interface accompanying complex formation.

    PubMed

    Chakravarty, Devlina; Janin, Joël; Robert, Charles H; Chakrabarti, Pinak

    2015-11-01

    Protein interactions are essential in all biological processes. The changes brought about in the structure when a free component forms a complex with another molecule need to be characterized for a proper understanding of molecular recognition as well as for the successful implementation of docking algorithms. Here, unbound (U) and bound (B) forms of protein structures from the Protein-Protein Interaction Affinity Database are compared in order to enumerate the changes that occur at the interface atoms/residues in terms of the solvent-accessible surface area (ASA), secondary structure, temperature factors (B factors) and disorder-to-order transitions. It is found that the interface atoms optimize contacts with the atoms in the partner protein, which leads to an increase in their ASA in the bound interface in the majority (69%) of the proteins when compared with the unbound interface, and this is independent of the root-mean-square deviation between the U and B forms. Changes in secondary structure during the transition indicate a likely extension of helices and strands at the expense of turns and coils. A reduction in flexibility during complex formation is reflected in the decrease in B factors of the interface residues on going from the U form to the B form. There is, however, no distinction in flexibility between the interface and the surface in the monomeric structure, thereby highlighting the potential problem of using B factors for the prediction of binding sites in the unbound form for docking another protein. 16% of the proteins have missing (disordered) residues in the U form which are observed (ordered) in the B form, mostly with an irregular conformation; the data set also shows differences in the composition of interface and non-interface residues in the disordered polypeptide segments as well as differences in their surface burial.

  7. [Nonstructural protein 1 of tick-borne encephalitis virus activates the expression of immunoproteasome subunits].

    PubMed

    Kuzmenko, Y V; Starodubova, E S; Karganova, G G; Timofeev, A V; Karpov, V L

    2016-01-01

    The interaction of viral proteins with host cell components plays an important role in antiviral immune response. One of the key steps of antiviral defense is the formation of immunoproteasomes. The effect of nonstructural protein 1 (NS1) of tick-borne encephalitis virus on the immunoproteasome formation was studied. It was shown that cell expression of NS1 does not reduce the efficacy of the immunoproteasome generation in response to interferon-γ stimulation and even increases the content of the immunoproteasome subunits without the interferon-γ treatment. Thus, NS1 of tick-borne encephalitis virus activates, rather than blocks the mechanisms of immune defense in the cell.

  8. Elg1, the major subunit of an alternative RFC complex, interacts with SUMO-processing proteins.

    PubMed

    Parnas, Oren; Amishay, Rona; Liefshitz, Batia; Zipin-Roitman, Adi; Kupiec, Martin

    2011-09-01

    PCNA is a homotrimeric ring with important roles in DNA replication and repair. PCNA is loaded and unloaded by the RFC complex, which is composed of five subunits (Rfc1-5). Three additional complexes that share with RFC the small subunits (Rfc2-5) and contain alternative large subunits were found in yeast and other eukaryotes. We have recently reported that one of these, the Elg1-RFC complex, interacts with SUMOylated PCNA and may play a role in its unloading during DNA repair. Here we report that a yeast-two-hybrid screen with the N terminus of Elg1(which interacts with SUMOylated PCNA) uncovered interactions with proteins that belong to the SUMO pathway, including Slx5 and Slx8, which form an E3 ubiquitin ligase that ubiquitinates SUMOylated proteins. Mutations in SLX5 result in a genomic instability phenotype similar to that of elg1 mutants. The physical interaction between the N terminus of Elg1 and Slx5 is mediated by poly-SUMO chains but not by PCNA modifications, and requires Siz2, but not Siz1, activity. Thus our results highlight the many important roles played by Elg1, some of which are PCNA-dependent and some PCNA-independent.

  9. An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse

    PubMed Central

    Almedom, Ruta B; Liewald, Jana F; Hernando, Guillermina; Schultheis, Christian; Rayes, Diego; Pan, Jie; Schedletzky, Thorsten; Hutter, Harald; Bouzat, Cecilia; Gottschalk, Alexander

    2009-01-01

    Nicotinic acetylcholine receptors (nAChRs) are homo- or heteropentameric ligand-gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell-surface expression and functional properties in Caenorhabditis elegans muscle. Loss of either type I TM protein, NRA-2 or NRA-4 (nicotinic receptor associated), affects two different types of muscle nAChRs and causes in vivo resistance to cholinergic agonists. Sensitivity to subtype-specific agonists of these nAChRs is altered differently, as demonstrated by whole-cell voltage-clamp of dissected adult muscle, when applying exogenous agonists or after photo-evoked, channelrhodopsin-2 (ChR2) mediated acetylcholine (ACh) release, as well as in single-channel recordings in cultured embryonic muscle. These data suggest that nAChRs desensitize faster in nra-2 mutants. Cell-surface expression of different subunits of the ‘levamisole-sensitive' nAChR (L-AChR) is differentially affected in the absence of NRA-2 or NRA-4, suggesting that they control nAChR subunit composition or allow only certain receptor assemblies to leave the ER. PMID:19609303

  10. Structure–function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring

    PubMed Central

    Schwer, Beate; Kruchten, Joshua; Shuman, Stewart

    2016-01-01

    A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure–function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein–RNA and protein–protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not. PMID:27417296

  11. Functional characterisation of the regulatory subunit of cyclic AMP-dependent protein kinase A homologue of Giardia lamblia: Differential expression of the regulatory and catalytic subunits during encystation.

    PubMed

    Gibson, Candace; Schanen, Brian; Chakrabarti, Debopam; Chakrabarti, Ratna

    2006-06-01

    To understand the functional roles of protein kinase A (PKA) during vegetative and differentiating states of Giardia parasites, we studied the structural and functional characteristics of the regulatory subunit of PKA (gPKAr) and its involvement in the giardial encystment process. Molecular cloning and characterisation showed that gPKAr contains two tandem 3'5'-cyclic adenosine monphosphate (cyclic AMP) binding domains at the C-terminal end and the interaction domain for the catalytic subunit. A number of consensus residues including in vivo phosphorylation site for PKAc and dimerisation/docking domain are present in gPKAr. The regulatory subunit physically interacts with the catalytic subunit and inhibits its kinase activity in the absence of cyclic AMP, which could be partially restored upon addition of cyclic AMP. Western blot analysis showed a marked reduction in the endogenous gPKAr concentration during differentiation of Giardia into cysts. An increased activity of gPKAc was also detected during encystation without any significant change in the protein concentration. Distinct localisations of gPKAc to the anterior flagella, basal bodies and caudal flagella as noted in trophozoites were absent in encysting cells at later stages. Instead, PKAc staining was punctate and located mostly to the cell periphery. Our study indicates possible enrichment of the active gPKAc during late stages of encystation, which may have implications in completion of the encystment process or priming of cysts for efficient excystation.

  12. Changes in protein structure at the interface accompanying complex formation

    PubMed Central

    Chakravarty, Devlina; Janin, Joël; Robert, Charles H.; Chakrabarti, Pinak

    2015-01-01

    Protein interactions are essential in all biological processes. The changes brought about in the structure when a free component forms a complex with another molecule need to be characterized for a proper understanding of molecular recognition as well as for the successful implementation of docking algorithms. Here, unbound (U) and bound (B) forms of protein structures from the Protein–Protein Interaction Affinity Database are compared in order to enumerate the changes that occur at the interface atoms/residues in terms of the solvent-accessible surface area (ASA), secondary structure, temperature factors (B factors) and disorder-to-order transitions. It is found that the interface atoms optimize contacts with the atoms in the partner protein, which leads to an increase in their ASA in the bound interface in the majority (69%) of the proteins when compared with the unbound interface, and this is independent of the root-mean-square deviation between the U and B forms. Changes in secondary structure during the transition indicate a likely extension of helices and strands at the expense of turns and coils. A reduction in flexibility during complex formation is reflected in the decrease in B factors of the interface residues on going from the U form to the B form. There is, however, no distinction in flexibility between the interface and the surface in the monomeric structure, thereby highlighting the potential problem of using B factors for the prediction of binding sites in the unbound form for docking another protein. 16% of the proteins have missing (disordered) residues in the U form which are observed (ordered) in the B form, mostly with an irregular conformation; the data set also shows differences in the composition of interface and non-interface residues in the disordered polypeptide segments as well as differences in their surface burial. PMID:26594372

  13. Molecular model of the G protein. alpha. subunit based on the crystal structure of the HRAS protein

    SciTech Connect

    Holbrook, S.R.; Kim, Sunghou )

    1989-03-01

    A structural model of guanine nucleotide-binding regulatory protein {alpha} subunits (G{sub {alpha}} subunits) is proposed based on the crystal structure of the catalytic domain of the human HRAS protein (p21{sup ras}). Because of low overall sequence similarity, structural and functional constraints were used to align the G{sub {alpha}} consensus sequence with that of p21{sup ras}. The resulting G{sub {alpha}} model specifies the spatial relationship among the guanine nucleotide-binding site, the binding site of the {beta}{gamma} subunit complex, likely regions of effector and receptor interaction, and sites of cholera and pertussis toxin modification. The locations in the model of the experimentally determined sites of proteolytic digestion, point mutation, monoclonal antibody binding, and toxin modification are consistent with and help explain the observed biological activity. Two important findings from our model are (i) the orientation of the G{sub {alpha}} model with respect to the membrane and (ii) the identification of the spatial proximity of the N- and C-terminal regions. Furthermore, by analogy to p21{sup ras}, the model assigns specific residues in G{sub {alpha}} required for binding the guanosine (G-box) and phosphates (PO{sub 4}-box) and identifies residues potentially involved in the conformational switch mechanism (S-box). Specification of these critical regions in the G{sub {alpha}} model suggests guidelines for construction of mutants and chimeric proteins to experimentally test structural and functional hypotheses.

  14. Mining the characteristic interaction patterns on protein-protein binding interfaces.

    PubMed

    Li, Yan; Liu, Zhihai; Han, Li; Li, Chengke; Wang, Renxiao

    2013-09-23

    Protein-protein interactions are observed in various biological processes. They are important for understanding the underlying molecular mechanisms and can be potential targets for developing small-molecule regulators of such processes. Previous studies suggest that certain residues on protein-protein binding interfaces are "hot spots". As an extension to this concept, we have developed a residue-based method to identify the characteristic interaction patterns (CIPs) on protein-protein binding interfaces, in which each pattern is a cluster of four contacting residues. Systematic analysis was conducted on a nonredundant set of 1,222 protein-protein binding interfaces selected out of the entire Protein Data Bank. Favored interaction patterns across different protein-protein binding interfaces were retrieved by considering both geometrical and chemical conservations. As demonstrated on two test tests, our method was able to predict hot spot residues on protein-protein binding interfaces with good recall scores and acceptable precision scores. By analyzing the function annotations and the evolutionary tree of the protein-protein complexes in our data set, we also observed that protein-protein interfaces sharing common characteristic interaction patterns are normally associated with identical or similar biological functions.

  15. Subunit exchange of polydisperse proteins: mass spectrometry reveals consequences of alphaA-crystallin truncation.

    PubMed

    Aquilina, J Andrew; Benesch, Justin L P; Ding, Lin Lin; Yaron, Orna; Horwitz, Joseph; Robinson, Carol V

    2005-04-15

    The small heat shock protein, alpha-crystallin, plays a key role in maintaining lens transparency by chaperoning structurally compromised proteins. This is of particular importance in the human lens, where proteins are exposed to post-translational modifications over the life-time of an individual. Here, we examine the structural and functional consequences of one particular modification of alphaA-crystallin involving the truncation of 5 C-terminal residues (alphaA(1-168)). Using novel mass spectrometry approaches and established biophysical techniques, we show that alphaA(1-168) forms oligomeric assemblies with a lower average molecular mass than wild-type alphaA-crystallin (alphaA(WT)). Also apparent from the mass spectra of both alphaA(WT) and alphaA(1-168) assemblies is the predominance of oligomers containing even numbers of subunits; interestingly, this preference is more marked for alphaA(1-168). To examine the rate of exchange of subunits between assemblies, we mixed alphaB crystallin with either alphaA(WT) or alphaA(1-168) and monitored in a real-time mass spectrometry experiment the formation of heteroligomers. The results show that there is a significant decrease in the rate of exchange when alphaA(1-168) is involved. These reduced exchange kinetics, however, have no effect upon chaperone efficiency, which is found to be closely similar for both alphaA(WT) and alphaA(1-168). Overall, therefore, our results allow us to conclude that, in contrast to mechanisms established for analogous proteins from plants, yeast, and bacteria, the rate of subunit exchange is not the critical parameter in determining efficient chaperone behavior for mammalian alphaA-crystallin.

  16. Differential association of protein subunits with the human RNase MRP and RNase P complexes.

    PubMed

    Welting, Tim J M; Kikkert, Bastiaan J; van Venrooij, Walther J; Pruijn, Ger J M

    2006-07-01

    RNase MRP is a eukaryotic endoribonuclease involved in nucleolar and mitochondrial RNA processing events. RNase MRP is a ribonucleoprotein particle, which is structurally related to RNase P, an endoribonuclease involved in pre-tRNA processing. Most of the protein components of RNase MRP have been reported to be associated with RNase P as well. In this study we determined the association of these protein subunits with the human RNase MRP and RNase P particles by glycerol gradient sedimentation and coimmunoprecipitation. In agreement with previous studies, RNase MRP sedimented at 12S and 60-80S. In contrast, only a single major peak was observed for RNase P at 12S. The analysis of individual protein subunits revealed that hPop4 (also known as Rpp29), Rpp21, Rpp20, and Rpp25 only sedimented in 12S fractions, whereas hPop1, Rpp40, Rpp38, and Rpp30 were also found in 60-80S fractions. In agreement with their cosedimentation with RNase P RNA in the 12S peak, coimmunoprecipitation with VSV-epitope-tagged protein subunits revealed that hPop4, Rpp21, and in addition Rpp14 preferentially associate with RNase P. These data show that hPop4, Rpp21, and Rpp14 may not be associated with RNase MRP. Furthermore, Rpp20 and Rpp25 appear to be associated with only a subset of RNase MRP particles, in contrast to hPop1, Rpp40, Rpp38, and Rpp30 (and possibly also hPop5), which are probably associated with all RNase MRP complexes. Our data are consistent with a transient association of Rpp20 and Rpp25 with RNase MRP, which may be inversely correlated to its involvement in pre-rRNA processing.

  17. Inhibition of the catalytic subunit of cAMP-dependent protein kinase by dicyclohexylcarbodiimide

    SciTech Connect

    Toner-Webb, J.; Taylor, S.S.

    1987-11-17

    The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) has been shown to inhibit the catalytic (C) subunit of adenosine cyclic 3',5'-phosphate dependent protein kinase in a time-dependent, irreversible manner. The rate of inactivation was first order and showed saturation kinetics with an apparent K/sub i/ of 60 ..mu..M. Magnesium adenosine 5'-triphosphate (MgATP) was capable of protecting against this inhibition, whereas neither a synthetic peptide substrate nor histone afforded protection. Mg alone afforded some protection. When the catalytic subunit was aggregated with the regulatory subunit in the holoenzyme complex, no inhibition was observed. The inhibition was enhanced at low pH, suggesting that a carboxylic acid group was the target for interaction with DCCD. On the basis of the protection studies, it is most likely that this carboxylic acid group is associated with the MgATP binding site, perhaps serving as a ligand for the metal. Efforts to identify the site that was modified by DCCD were made. In no case was radioactivity incorporated into the protein, suggesting that the irreversible inhibition was due to an intramolecular cross-link between a reactive carboxylic acid group and a nearby amino group. Differential peptide mapping identified a single peptide that was consistently lost as a consequence of DCCD inhibition. This peptide (residues 166-189) contained four carboxylic acid residues as well as an internal Lys. Two of these carboxyl groups, Asp-166 and Asp-184, are conserved in all protein kinases, including oncogene transforming proteins and growth factor receptors, and thus are likely to play an essential role.

  18. Pheromone action regulates G-protein alpha-subunit myristoylation in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Dohlman, H G; Goldsmith, P; Spiegel, A M; Thorner, J

    1993-01-01

    Myristic acid (C14:0) is added to the N-terminal glycine residue of the alpha subunits of certain receptor-coupled guanine nucleotide-binding regulatory proteins (G proteins). The G alpha subunit (GPA1 gene product) coupled to yeast pheromone receptors exists as a pool of both myristoylated and unmyristolyated species. After treatment of MATa cells with alpha factor, the myristoylated form of Gpa1p increases dramatically, and the unmyristoylated form decreases concomitantly. This pheromone-stimulated shift depends on the function of STE2 (alpha-factor receptor), STE11 (a protein kinase in the response pathway), and NMT1 (myristoyl-CoA:protein N-myristoyltransferase) genes and uses the existing pool of fatty acids (is not blocked by cerulenin). Myristoylated Gpa1p persists long after pheromone is removed. Because myristoylation is essential for proper G alpha-G beta gamma association and receptor coupling, pheromone-dependent stimulation of Gpa1p myristoylation may be an important contributing factor in adaptation after signal transmission. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8415763

  19. PRKACA: the catalytic subunit of protein kinase A and adrenocortical tumors

    PubMed Central

    Berthon, Annabel S.; Szarek, Eva; Stratakis, Constantine A.

    2015-01-01

    Cyclic-AMP (cAMP)-dependent protein kinase (PKA) is the main effector of cAMP signaling in all tissues. Inactivating mutations of the PRKAR1A gene, coding for the type 1A regulatory subunit of PKA, are responsible for Carney complex and primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A inactivation and PKA dysregulation have been implicated in various types of adrenocortical pathologies associated with ACTH-independent Cushing syndrome (AICS) from PPNAD to adrenocortical adenomas and cancer, and other forms of bilateral adrenocortical hyperplasias (BAH). More recently, mutations of PRKACA, the gene coding for the catalytic subunit C alpha (Cα), were also identified in the pathogenesis of adrenocortical tumors. PRKACA copy number gain was found in the germline of several patients with cortisol-producing BAH, whereas the somatic Leu206Arg (c.617A>C) recurrent PRKACA mutation was found in as many as half of all adrenocortical adenomas associated with AICS. In vitro analysis demonstrated that this mutation led to constitutive Cα activity, unregulated by its main partners, the PKA regulatory subunits. In this review, we summarize the current understanding of the involvement of PRKACA in adrenocortical tumorigenesis, and our understanding of PKA's role in adrenocortical lesions. We also discuss potential therapeutic advances that can be made through targeting of PRKACA and the PKA pathway. PMID:26042218

  20. Modeling of the water network at protein-RNA interfaces.

    PubMed

    Li, Yiyu; Sutch, Brian T; Bui, Huynh-Hoa; Gallaher, Timothy K; Haworth, Ian S

    2011-06-27

    Water plays an important role in the mediation of biomolecular interactions. Thus, accurate prediction and evaluation of water-mediated interactions is an important element in the computational design of interfaces involving proteins, RNA, and DNA. Here, we use an algorithm (WATGEN) to predict the locations of interfacial water molecules for a data set of 224 protein-RNA interfaces. The accuracy of the prediction is validated against water molecules present in the X-ray structures of 105 of these complexes. The complexity of the water networks is deconvoluted through definition of the characteristics of each water molecule based on its bridging properties between the protein and RNA and on its depth in the interface with respect to the bulk solvent. This approach has the potential for scoring the water network for incorporation into the computational design of protein-RNA complexes.

  1. Agonist-specific conformational changes in the α1-γ2 subunit interface of the GABA A receptor.

    PubMed

    Eaton, Megan M; Lim, You Bin; Bracamontes, John; Steinbach, Joe Henry; Akk, Gustav

    2012-08-01

    The GABA(A) receptor undergoes conformational changes upon the binding of agonist that lead to the opening of the channel gate and a flow of small anions across the cell membrane. Besides the transmitter GABA, allosteric ligands such as the general anesthetics pentobarbital and etomidate can activate the receptor. Here, we have investigated the agonist specificity of structural changes in the extracellular domain of the receptor. We used the substituted cysteine accessibility method and focused on the γ2(S195C) site (loop F). We show that modification of the site with (2-sulfonatoethyl)methanethiosulfonate (MTSES) results in an enhanced response to GABA, indicating accessibility of the resting receptor to the modifying agent. Coapplication of GABA or muscimol, but not of gabazine, with MTSES prevented the effect, suggesting that GABA and muscimol elicit a conformational change that reduces access to the γ2(S195C) site. Exposure of the receptors to MTSES in the presence of the allosteric activators pentobarbital and etomidate resulted in an enhanced current response indicating accessibility and labeling of the γ2(S195C) site. However, comparison of the rates of modification indicated that labeling in the presence of etomidate was significantly faster than that in the presence of pentobarbital or gabazine or in resting receptors. We infer from the data that the structure of the α1-γ2 subunit interface undergoes agonist-specific conformational changes.

  2. Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae.

    PubMed

    Lee, Laura J Y; Klute, Mary J; Herman, Emily K; Read, Betsy; Dacks, Joel B

    2015-11-01

    The phylum Haptophyta (Diaphoratickes) contains marine algae that perform biomineralization, extruding large, distinctive calcium carbonate scales (coccoliths) that completely cover the cell. Coccolith production is an important part of global carbon cycling; however, the membrane trafficking pathway by which they are secreted has not yet been elucidated. In most eukaryotes, post-Golgi membrane trafficking involves five heterotetrameric adaptor protein (AP) complexes, which impart cargo selection specificity. To better understand coccolith secretion, we performed comparative genomic, phylogenetic, and transcriptomic analyses of the AP complexes in Emiliania huxleyi strains 92A, Van556, EH2, and CCMP1516, and related haptophytes Gephyrocapsa oceanica and Isochrysis galbana; the latter has lost the ability to biomineralize. We show that haptophytes have a modified membrane trafficking system (MTS), as we found both AP subunit losses and duplications. Additionally, we identified a single conserved subunit of the AP-related TSET complex, whose expression suggests a functional role in membrane trafficking. Finally, we detected novel alpha adaptin ear and gamma adaptin ear proteins, the first of their kind to be described outside of opisthokonts. These novel ear proteins and the sculpting of the MTS may support the capacity for biomineralization in haptophytes, enhancing their ability to perform this highly specialized form of secretion.

  3. Crystal structure of truncated human coatomer protein complex subunit ζ1 (Copζ1).

    PubMed

    Lunev, Sergey; Semmelink, Marije F W; Xian, Jia Ling; Ma, Kai Yu; Leenders, Anna J A; Dömling, Alexander S S; Shtutman, Michael; Groves, Matthew R

    2017-01-01

    The majority of modern anticancer approaches target DNA/protein targets involved in tumour-cell proliferation. Such approaches have a major drawback, as nonproliferating cancer cells remain unaffected and may cause relapse or remission. Human coatomer protein complex I (COPI) subunit ζ (Copζ), a component of the coat protein involved in cell apoptosis and intracellular trafficking, has recently been proposed as a potential anticancer drug target. Previous studies have shown that two different isoforms of the Copζ subunit exist in mammalian cells. While normal cells express both Copζ1 and Copζ2 isoforms, various types of tumour cells display a loss of Copζ2 expression and rely solely on Copζ1 for growth and survival. Subsequent knockdown of Copζ1 results in specific inhibition of both proliferating and dormant tumour-cell populations, with no adverse growth effects on normal cells. Therefore, a Copζ1-targeting therapy was proposed to bypass the problem of dormant cancer cells that are resistant to conventional antiproliferative drugs, which is the major cause of tumour relapse. In order to aid in structure-based inhibitor design, a crystal structure is required. In this article, the recombinant expression, purification, crystallization and crystal structure of Copζ1, as well as the expression and purification of Copζ2, are reported.

  4. Chemical modulators working at pharmacological interface of target proteins.

    PubMed

    Jeon, Young Ho; Lee, Jin Young; Kim, Sunghoon

    2012-03-15

    For last few decades, the active site cleft and substrate-binding site of enzymes as well as ligand-binding site of the receptors have served as the main pharmacological space for drug discovery. However, rapid accumulation of proteome and protein network analysis data has opened a new therapeutic space that is the interface between the interacting proteins. Due to the complexity of the interaction modes and the numbers of the participating components, it is still challenging to identify the chemicals that can accurately control the protein-protein interactions at desire. Nonetheless, the number of chemical drugs and candidates working at the interface of the interacting proteins are rapidly increasing. This review addresses the current case studies and state-of-the-arts in the development of small chemical modulators controlling the interactions of the proteins that have pathological implications in various human diseases such as cancer, immune disorders, neurodegenerative and infectious diseases.

  5. Rice Ribosomal Protein Large Subunit Genes and Their Spatio-temporal and Stress Regulation

    PubMed Central

    Moin, Mazahar; Bakshi, Achala; Saha, Anusree; Dutta, Mouboni; Madhav, Sheshu M.; Kirti, P. B.

    2016-01-01

    Ribosomal proteins (RPs) are well-known for their role in mediating protein synthesis and maintaining the stability of the ribosomal complex, which includes small and large subunits. In the present investigation, in a genome-wide survey, we predicted that the large subunit of rice ribosomes is encoded by at least 123 genes including individual gene copies, distributed throughout the 12 chromosomes. We selected 34 candidate genes, each having 2–3 identical copies, for a detailed characterization of their gene structures, protein properties, cis-regulatory elements and comprehensive expression analysis. RPL proteins appear to be involved in interactions with other RP and non-RP proteins and their encoded RNAs have a higher content of alpha-helices in their predicted secondary structures. The majority of RPs have binding sites for metal and non-metal ligands. Native expression profiling of 34 ribosomal protein large (RPL) subunit genes in tissues covering the major stages of rice growth shows that they are predominantly expressed in vegetative tissues and seedlings followed by meiotically active tissues like flowers. The putative promoter regions of these genes also carry cis-elements that respond specifically to stress and signaling molecules. All the 34 genes responded differentially to the abiotic stress treatments. Phytohormone and cold treatments induced significant up-regulation of several RPL genes, while heat and H2O2 treatments down-regulated a majority of them. Furthermore, infection with a bacterial pathogen, Xanthomonas oryzae, which causes leaf blight also induced the expression of 80% of the RPL genes in leaves. Although the expression of RPL genes was detected in all the tissues studied, they are highly responsive to stress and signaling molecules indicating that their encoded proteins appear to have roles in stress amelioration besides house-keeping. This shows that the RPL gene family is a valuable resource for manipulation of stress tolerance in

  6. Dynamic Nuclear Polarization of membrane proteins: covalently bound spin-labels at protein-protein interfaces

    PubMed Central

    Wylie, Benjamin J; Dzikovski, Boris G.; Pawsey, Shane; Caporini, Marc; Rosay, Melanie; Freed, Jack H.; McDermott, Ann E.

    2016-01-01

    We demonstrate that dynamic nuclear polarization (DNP) of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of 6-fold for the dimeric protein. The enhancement affect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces. PMID:25828256

  7. The nano-plasma interface: Implications of the protein corona.

    PubMed

    Wolfram, Joy; Yang, Yong; Shen, Jianliang; Moten, Asad; Chen, Chunying; Shen, Haifa; Ferrari, Mauro; Zhao, Yuliang

    2014-12-01

    The interactions between nanoparticles and macromolecules in the blood plasma dictate the biocompatibility and efficacy of nanotherapeutics. Accordingly, the properties of nanoparticles and endogenous biomolecules change at the nano-plasma interface. Here, we review the implications of such changes including toxicity, immunological recognition, molecular targeting, biodistribution, intracellular uptake, and drug release. Although this interface poses several challenges for nanomedicine, it also presents opportunities for exploiting nanoparticle-protein interactions.

  8. Protein interfaces and intersubunit bonding. The case of tomato bushy stunt virus.

    PubMed Central

    Harrison, S C

    1980-01-01

    An atomic model of the subunit of tomato bushy stunt virus (TBSV) has been constructed to fit an electron density map at 2.9 A resolution. Subunit interfaces show networks of polar residues forming H-bonds and salt bridges. The way in which alternative specific bonding geometries are built into a contact are described. PMID:7248446

  9. Acid-base chemistry of frustrated water at protein interfaces.

    PubMed

    Fernández, Ariel

    2016-01-01

    Water molecules at a protein interface are often frustrated in hydrogen-bonding opportunities due to subnanoscale confinement. As shown, this condition makes them behave as a general base that may titrate side-chain ammonium and guanidinium cations. Frustration-based chemistry is captured by a quantum mechanical treatment of proton transference and shown to remove same-charge uncompensated anticontacts at the interface found in the crystallographic record and in other spectroscopic information on the aqueous interface. Such observations are untenable within classical arguments, as hydronium is a stronger acid than ammonium or guanidinium. Frustration enables a directed Grotthuss mechanism for proton transference stabilizing same-charge anticontacts.

  10. Type B Heterotrimeric G Protein γ-Subunit Regulates Auxin and ABA Signaling in Tomato[OPEN

    PubMed Central

    Subramaniam, Gayathery; Trusov, Yuri; Hayashi, Satomi; Batley, Jacqueline

    2016-01-01

    Heterotrimeric G proteins composed of α, β, and γ subunits are central signal transducers mediating the cellular response to multiple stimuli in most eukaryotes. Gγ subunits provide proper cellular localization and functional specificity to the heterotrimer complex. Plant Gγ subunits, divided into three structurally distinct types, are more diverse than their animal counterparts. Type B Gγ subunits, lacking a carboxyl-terminal isoprenylation motif, are found only in flowering plants. We present the functional characterization of type B Gγ subunit (SlGGB1) in tomato (Solanum lycopersicum). We show that SlGGB1 is the most abundant Gγ subunit in tomato and strongly interacts with the Gβ subunit. Importantly, the green fluorescent protein-SlGGB1 fusion protein as well as the carboxyl-terminal yellow fluorescent protein-SlGGB1/amino-terminal yellow fluorescent protein-Gβ heterodimer were localized in the plasma membrane, nucleus, and cytoplasm. RNA interference-mediated silencing of SlGGB1 resulted in smaller seeds, higher number of lateral roots, and pointy fruits. The silenced lines were hypersensitive to exogenous auxin, while levels of endogenous auxins were lower or similar to those of the wild type. SlGGB1-silenced plants also showed strong hyposensitivity to abscisic acid (ABA) during seed germination but not in other related assays. Transcriptome analysis of the transgenic seeds revealed abnormal expression of genes involved in ABA sensing, signaling, and response. We conclude that the type B Gγ subunit SlGGB1 mediates auxin and ABA signaling in tomato. PMID:26668332

  11. Design of protein function leaps by directed domain interface evolution

    PubMed Central

    Huang, Jin; Koide, Akiko; Makabe, Koki; Koide, Shohei

    2008-01-01

    Most natural proteins performing sophisticated tasks contain multiple domains where an active site is located at the domain interface. Comparative structural analyses suggest that major leaps in protein function occur through gene recombination events that connect two or more protein domains to generate a new active site, frequently occurring at the newly created domain interface. However, such functional leaps by combination of unrelated domains have not been directly demonstrated. Here we show that highly specific and complex protein functions can be generated by joining a low-affinity peptide-binding domain with a functionally inert second domain and subsequently optimizing the domain interface. These directed evolution processes dramatically enhanced both affinity and specificity to a level unattainable with a single domain, corresponding to >500-fold and >2,000-fold increases of affinity and specificity, respectively. An x-ray crystal structure revealed that the resulting “affinity clamp” had clamshell architecture as designed, with large additional binding surface contributed by the second domain. The affinity clamps having a single-nanomolar dissociation constant outperformed a monoclonal antibody in immunochemical applications. This work establishes evolutionary paths from isolated domains with primitive function to multidomain proteins with sophisticated function and introduces a new protein-engineering concept that allows for the generation of highly functional affinity reagents to a predefined target. The prevalence and variety of natural interaction domains suggest that numerous new functions can be designed by using directed domain interface evolution. PMID:18445649

  12. Magnesium chelatase subunit D from pea: characterization of the cDNA, heterologous expression of an enzymatically active protein and immunoassay of the native protein.

    PubMed

    Luo, M; Weinstein, J D; Walker, C J

    1999-12-01

    Mg-chelatase catalyzes the insertion of Mg into protoporphyrin and lies at the branchpoint of heme and (bacterio)chlorophyll synthesis. In prokaryotes, three genes--BchI, D and H--encode subunits for Mg-chelatase. In higher plants, homologous cDNAs for the I, D and H subunits have been characterized. Since the N-terminal half of the D subunit is homologous to the I subunit, the C-terminal portion of the pea D was used for antigen production. The antibody recognized the chloroplast D subunit and was used to demonstrate that this subunit associated with the membranes in the presence of MgCl2. The antibody immunoprecipitated the native protein and inhibited Mg-chelatase activity. Expression in Escherichia coli with a construct for the full-length protein (minus the putative transit peptide) resulted in induction of 24.5 kDa (major) and 89 kDa (minor) proteins which could only be solubilized in 6 M urea. However, when host cells were co-transformed with expression vectors for the full-length D subunit and for the 70 kDa HSP chaperonin protein, a substantial portion of the 89 kDa protein was expressed in a soluble form which was active in a Mg-chelatase reconstitution assay.

  13. RNase P protein subunit Rpp29 represses histone H3.3 nucleosome deposition

    PubMed Central

    Newhart, Alyshia; Powers, Sara Lawrence; Shastrula, Prashanth Krishna; Sierra, Isabel; Joo, Lucy M.; Hayden, James E.; Cohen, Andrew R.; Janicki, Susan M.

    2016-01-01

    In mammals, histone H3.3 is a critical regulator of transcription state change and heritability at both euchromatin and heterochromatin. The H3.3-specific chaperone, DAXX, together with the chromatin-remodeling factor, ATRX, regulates H3.3 deposition and transcriptional silencing at repetitive DNA, including pericentromeres and telomeres. However, the events that precede H3.3 nucleosome incorporation have not been fully elucidated. We previously showed that the DAXX-ATRX-H3.3 pathway regulates a multi-copy array of an inducible transgene that can be visualized in single living cells. When this pathway is impaired, the array can be robustly activated. H3.3 is strongly recruited to the site during activation where it accumulates in a complex with transcribed sense and antisense RNA, which is distinct from the DNA/chromatin. This suggests that transcriptional events regulate H3.3 recruited to its incorporation sites. Here we report that the nucleolar RNA proteins Rpp29, fibrillarin, and RPL23a are also components of this H3.3/RNA complex. Rpp29 is a protein subunit of RNase P. Of the other subunits, POP1 and Rpp21 are similarly recruited suggesting that a variant of RNase P regulates H3.3 chromatin assembly. Rpp29 knockdown increases H3.3 chromatin incorporation, which suggests that Rpp29 represses H3.3 nucleosome deposition, a finding with implications for epigenetic regulation. PMID:26842893

  14. Activity, Expression and Function of a Second Drosophila Protein Kinase a Catalytic Subunit Gene

    PubMed Central

    Melendez, A.; Li, W.; Kalderon, D.

    1995-01-01

    The DC2 gene was isolated previously on the basis of sequence similarity to DCO, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 did not affect the viability or phenotype of imaginal disc cells lacking DC0 activity or embryonic hatching of animals with reduced DC0 activity. Furthermore, transgenes expressing DC2 from a DC0 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. PMID:8601490

  15. Expression of the G-protein alpha-subunit gustducin in mammalian spermatozoa.

    PubMed

    Fehr, Johanna; Meyer, Dorke; Widmayer, Patricia; Borth, Heike Claudia; Ackermann, Frauke; Wilhelm, Beate; Gudermann, Thomas; Boekhoff, Ingrid

    2007-01-01

    Although chemotaxis has been proposed to guide sperm to egg throughout the animal kingdom, sperm attractants released from mammalian eggs have not been identified. Since the G protein subunit alpha-gustducin is accepted as a marker of chemosensitive cells, attempts were made to explore whether alpha-gustducin is also expressed in spermatozoa of mammals. Immunohistochemical approaches using an anti-alpha-gustducin-specific antibody revealed the most intense immunoreactivity in differentiating spermatids. Further evidence for the alpha-gustducin expression was obtained analyzing testicular and sperm-derived tissue preparations in western blot analyses. To elucidate whether alpha-gustducin is retained in mature spermatozoa, epididymal mouse and rat sperm were subjected to immunocytochemistry as well as immunogold electron microscopy. A specific staining was obtained within the circumference of the midpiece-localized mitochondria, on the axoneme and the outer dense fibers surrounding the microtubules of this region, whereas no labeling was detectable in the end piece regions. The analysis of ejaculated bovine and human sperm revealed a comparable segmental distribution pattern for alpha-gustducin. Although a possible function for alpha-gustducin has yet to be determined, the axonemal-associated localization within the midpiece and principal piece of different mammalian spermatozoa raises the possibility that this G protein alpha-subunit may process intracellular signals controlling sperm motility.

  16. Activity, expression and function of a second Drosophila protein kinase a catalytic subunit gene

    SciTech Connect

    Melendez, A.; Li, W.; Kalderon, D.

    1995-12-01

    The DC2 was isolated previously on the basis of sequence similarity to DC0, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. 62 refs., 10 figs., 2 tabs.

  17. Moving Iron through Ferritin Protein Nanocages Depends on Residues throughout Each Four α-Helix Bundle Subunit*

    PubMed Central

    Haldar, Suranjana; Bevers, Loes E.; Tosha, Takehiko; Theil, Elizabeth C.

    2011-01-01

    Eukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based Fe2+/O2 oxidoreduction and formation of [Fe3+O]n multimers within the protein cage, en route to the cavity, at sites distributed over ∼50 Å. Recent NMR and Co2+-protein x-ray diffraction (XRD) studies identified the entire iron path and new metal-protein interactions: (i) lines of metal ions in 8 Fe2+ ion entry channels with three-way metal distribution points at channel exits and (ii) interior Fe3+O nucleation channels. To obtain functional information on the newly identified metal-protein interactions, we analyzed effects of amino acid substitution on formation of the earliest catalytic intermediate (diferric peroxo-A650 nm) and on mineral growth (Fe3+O-A350 nm), in A26S, V42G, D127A, E130A, and T149C. The results show that all of the residues influenced catalysis significantly (p < 0.01), with effects on four functions: (i) Fe2+ access/selectivity to the active sites (Glu130), (ii) distribution of Fe2+ to each of the three active sites near each ion channel (Asp127), (iii) product (diferric oxo) release into the Fe3+O nucleation channels (Ala26), and (iv) [Fe3+O]n transit through subunits (Val42, Thr149). Synthesis of ferritin biominerals depends on residues along the entire length of H subunits from Fe2+ substrate entry at 3-fold cage axes at one subunit end through active sites and nucleation channels, at the other subunit end, inside the cage at 4-fold cage axes. Ferritin subunit-subunit geometry contributes to mineral order and explains the physiological impact of ferritin H and L subunits. PMID:21592958

  18. A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export.

    PubMed

    Wild, Thomas; Horvath, Peter; Wyler, Emanuel; Widmann, Barbara; Badertscher, Lukas; Zemp, Ivo; Kozak, Karol; Csucs, Gabor; Lund, Elsebet; Kutay, Ulrike

    2010-10-26

    The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.

  19. SPLINTS: small-molecule protein ligand interface stabilizers.

    PubMed

    Fischer, Eric S; Park, Eunyoung; Eck, Michael J; Thomä, Nicolas H

    2016-04-01

    Regulatory protein-protein interactions are ubiquitous in biology, and small molecule protein-protein interaction inhibitors are an important focus in drug discovery. Remarkably little attention has been given to the opposite strategy-stabilization of protein-protein interactions, despite the fact that several well-known therapeutics act through this mechanism. From a structural perspective, we consider representative examples of small molecules that induce or stabilize the association of protein domains to inhibit, or alter, signaling for nuclear hormone, GTPase, kinase, phosphatase, and ubiquitin ligase pathways. These SPLINTS (small-molecule protein ligand interface stabilizers) drive interactions that are in some cases physiologically relevant, and in others entirely adventitious. The diverse structural mechanisms employed suggest approaches for a broader and systematic search for such compounds in drug discovery.

  20. Protein bodies from the cotyledons of Cytisus scoparius L. (Link). Ultrastructure, isolation, and subunit composition of albumin, legumin and vicilin.

    PubMed

    Citharel, L; Citharel, J

    1985-09-01

    The structure of protein bodies differs in the upper and lower parts of the cotyledons of mature seeds of Cytisus scoparius L. The palisade-mesophyll cells contain essentially homogeneous protein bodies, without globoids, but the protein bodies of the spongy-mesophyll cells are heterogeneous, with numerous globoids. Albumins, legumins and vicilins were selectively extracted from isolated protein bodies and their subunits separated by SDS-PAGE, under non-reducing and reducing conditions.

  1. Neutralizing monoclonal antibodies against ricin's enzymatic subunit interfere with protein disulfide isomerase-mediated reduction of ricin holotoxin in vitro.

    PubMed

    O'Hara, Joanne M; Mantis, Nicholas J

    2013-09-30

    The penultimate event in the intoxication of mammalian cells by ricin toxin is the reduction, in the endoplasmic reticulum (ER), of the intermolecular disulfide bond that links ricin's enzymatic (RTA) and binding (RTB) subunits. In this report we adapted an in vitro protein disulfide isomerase (PDI)-mediated reduction assay to test the hypothesis that the RTA-specific neutralizing monoclonal antibody (mAb) IB2 interferes with the liberation of RTA from RTB. IB2 recognizes an epitope located near the interface between RTA and RTB and, like a number of other RTA-specific neutralizing mAbs, is proposed to neutralize ricin intracellularly. In this study, we found that IB2 virtually eliminated the reduction of ricin holotoxin into RTA and RTB in vitro. Surprisingly, three other neutralizing mAbs (GD12, R70 and SyH7) that bind epitopes at considerable distance from ricin's disulfide bond were as effective (or nearly as effective) as IB2 in interfering with PDI-mediated liberation of RTA from RTB. By contrast, two non-neutralizing RTA-specific mAbs, FGA12 and SB1, did not affect PDI-mediated reduction of ricin. These data reveal a possible mechanism by which RTA-specific antibodies may neutralize ricin intracellularly, provided they are capable of trafficking in association with ricin from the cell surface to the ER.

  2. Domain-Opening and Dynamic Coupling in the α-Subunit of Heterotrimeric G Proteins

    PubMed Central

    Yao, Xin-Qiu; Grant, Barry J.

    2013-01-01

    Heterotrimeric G proteins are conformational switches that turn on intracellular signaling cascades in response to the activation of G-protein-coupled receptors. Receptor activation by extracellular stimuli promotes a cycle of GTP binding and hydrolysis on the G protein α-subunit (Gα). Important conformational transitions occurring during this cycle have been characterized from extensive crystallographic studies of Gα. However, the link between the observed conformations and the mechanisms involved in G-protein activation and effector interaction remain unclear. Here we describe a comprehensive principal component analysis of available Gα crystallographic structures supplemented with extensive unbiased conventional and accelerated molecular dynamics simulations that together characterize the response of Gα to GTP binding and hydrolysis. Our studies reveal details of activating conformational changes as well as the intrinsic flexibility of the α-helical domain that includes a large-scale 60° domain opening under nucleotide-free conditions. This result is consistent with the recently reported open crystal structure of Gs, the stimulatory G protein for adenylyl cyclase, in complex with the α2 adrenergic receptor. Sets of unique interactions potentially important for the conformational transition are also identified. Moreover simulations reveal nucleotide-dependent dynamical couplings of distal regions and residues potentially important for the allosteric link between functional sites. PMID:23870276

  3. Flexibility and small pockets at protein-protein interfaces: New insights into druggability.

    PubMed

    Jubb, Harry; Blundell, Tom L; Ascher, David B

    2015-10-01

    The transient assembly of multiprotein complexes mediates many aspects of cell regulation and signalling in living organisms. Modulation of the formation of these complexes through targeting protein-protein interfaces can offer greater selectivity than the inhibition of protein kinases, proteases or other post-translational regulatory enzymes using substrate, co-factor or transition state mimetics. However, capitalising on protein-protein interaction interfaces as drug targets has been hindered by the nature of interfaces that tend to offer binding sites lacking the well-defined large cavities of classical drug targets. In this review we posit that interfaces formed by concerted folding and binding (disorder-to-order transitions on binding) of one partner and other examples of interfaces where a protein partner is bound through a continuous epitope from a surface-exposed helix, flexible loop or chain extension may be more tractable for the development of "orthosteric", competitive chemical modulators; these interfaces tend to offer small-volume but deep pockets and/or larger grooves that may be bound tightly by small chemical entities. We discuss examples of such protein-protein interaction interfaces for which successful chemical modulators are being developed.

  4. Detection, characterization and quantitation of coxsackievirus A16 using polyclonal antibodies against recombinant capsid subunit proteins.

    PubMed

    Liu, Qingwei; Ku, Zhiqiang; Cai, Yicun; Sun, Bing; Leng, Qibin; Huang, Zhong

    2011-04-01

    Coxsackievirus A16 (CVA16), together with enterovirus type 71 (EV71), is responsible for most cases of hand, foot and mouth disease (HFMD) worldwide. Recent findings suggest that the recombination between CVA16 and EV71, and co-circulation of these two viruses may have contributed to the increase of HFMD cases in China over the past few years. Thus, for CVA16, further understanding of its virology, epidemiology and development of diagnostic tests and vaccines are of importance. The present study aimed to develop reagents and protocols for the detection, characterization and quantitation of CVA16. Recombinant CVA16 capsid subunit proteins VP0, VP3 and truncated VP1, were produced in Escherichia coli and used to immunize guinea pigs to generate polyclonal antibodies. The resultant three antisera detected specifically CVA16 propagated in Vero cells by immunostaining, ELISA and Western blotting. The antisera was used to show that CVA16 capsids were composed of correctly processed VP0, VP1 and VP3 subunits, and were present in the form of efficiently assembled particles. A method for the quantitation of the yield of CVA16 in Vero cells was established based on a Western blotting protocol using the recombinant VP0 as a reference standard and anti-VP0 as the detection antibody. This study shows the development and validation of reagents and methods, for qualitative and quantitative determination of CVA16, which are essential for the development of vaccines.

  5. AMP-activated protein kinase (AMPK) α2 subunit mediates glycolysis in postmortem skeletal muscle.

    PubMed

    Liang, Junfang; Yang, Qiyuan; Zhu, Mei-Jun; Jin, Ye; Du, Min

    2013-11-01

    Postmortem glycolysis is directly linked to the incidences of PSE (pale, soft and exudative) and DFD (dark, firm and dry) meats which cause significant loss to meat industry. AMP-activated protein kinase (AMPK) is a major regulator of postmortem glycolysis. However, there are two isoforms of the AMPKα catalytic subunit, and their roles in glycolysis of postmortem muscle remain unclear. The objective was to identify the isoform specific roles of AMPK in postmortem glycolysis. Wild type, AMPKα1, and AMPKα2 knockout (KO) mice were used in the current study. AMPK in Longissimus muscle was activated shortly after death. AMPKα2 but not AMPKα1 KO abolished the activity of AMPK in postmortem muscle. In addition, AMPKα2 KO reduced postmortem pH decline and the generation of lactate, while AMPKα1 KO had no significant effect. Finally, the glycogen content of skeletal muscle was reduced in AMPKα2 KO but not AMPKα1 KO mice. Data clearly demonstrate that AMPKα2 catalytic subunit mainly regulates postmortem glycolysis in muscle.

  6. Single Amino Acid Polymorphisms of Pertussis Toxin Subunit S2 (PtxB) Affect Protein Function

    PubMed Central

    Millen, Scott H.; Watanabe, Mineo; Komatsu, Eiji; Yamaguchi, Fuminori; Nagasawa, Yuki; Suzuki, Eri; Monaco, Haleigh; Weiss, Alison A.

    2015-01-01

    Whooping cough due to Bordetella pertussis is increasing in incidence, in part due to accumulation of mutations which increase bacterial fitness in highly vaccinated populations. Polymorphisms in the pertussis toxin, ptxA and ptxB genes, and the pertactin, prn genes of clinical isolates of Bordetella pertussis collected in Cincinnati from 1989 through 2005 were examined. While the ptxA and prn genotypes were variable, all 48 strains had the ptxB2 genotype; ptxB1 encodes glycine at amino acid 18 of the S2 subunit of pertussis toxin, while ptxB2 encodes serine. We investigated antigenic and functional differences of PtxB1 and PtxB2. The S2 protein was not very immunogenic. Only a few vaccinated or individuals infected with B. pertussis developed antibody responses to the S2 subunit, and these sera recognized both polymorphic forms equally well. Amino acid 18 of S2 is in a glycan binding domain, and the PtxB forms displayed differences in receptor recognition and toxicity. PtxB1 bound better to the glycoprotein, fetuin, and Jurkat T cells in vitro, but the two forms were equally effective at promoting CHO cell clustering. To investigate in vivo activity of Ptx, one μg of Ptx was administered to DDY mice and blood was collected on 4 days after injection. PtxB2 was more effective at promoting lymphocytosis in mice. PMID:26375454

  7. The Escherichia coli RNA polymerase alpha subunit and transcriptional activation by bacteriophage lambda CII protein.

    PubMed

    Gabig, M; Obuchowski, M; Ciesielska, A; Latała, B; Wegrzyn, A; Thomas, M S; Wegrzyn, G

    1998-01-01

    Bacteriophage lambda is not able to lysogenise the Escherichia coli rpoA341 mutant. This mutation causes a single amino acid substitution Lys271Glu in the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). Our previous studies indicated that the impaired lysogenisation of the rpoA341 host is due to a defect in transcriptional activation by the phage CII protein and suggested a role for alphaCTD in this process. Here we used a series of truncation and point mutants in the rpoA gene placed on a plasmid to investigate the process of transcriptional activation by the cII gene product. Our results indicate that amino-acid residues 265, 268 and 271 in the a subunit may play an important role in the CII-mediated activation of the pE promoter (most probably residue 271) or may be involved in putative interactions between alphaCTD and an UP-like element near pE (most probably residues 265 and 268). Measurement of the activity of pE-lacZ, pI-lacZ and p(aQ)-lacZ fusions in the rpoA+ and rpoA341 hosts demonstrated that the mechanism of activation of these CII-dependent promoters may be in each case different.

  8. The AMP-activated protein kinase α2 catalytic subunit controls whole-body insulin sensitivity

    PubMed Central

    Viollet, Benoit; Andreelli, Fabrizio; Jørgensen, Sebastian B.; Perrin, Christophe; Geloen, Alain; Flamez, Daisy; Mu, James; Lenzner, Claudia; Baud, Olivier; Bennoun, Myriam; Gomas, Emmanuel; Nicolas, Gaël; Wojtaszewski, Jørgen F.P.; Kahn, Axel; Carling, David; Schuit, Frans C.; Birnbaum, Morris J.; Richter, Erik A.; Burcelin, Rémy; Vaulont, Sophie

    2003-01-01

    AMP-activated protein kinase (AMPK) is viewed as a fuel sensor for glucose and lipid metabolism. To better understand the physiological role of AMPK, we generated a knockout mouse model in which the AMPKα2 catalytic subunit gene was inactivated. AMPKα2–/– mice presented high glucose levels in the fed period and during an oral glucose challenge associated with low insulin plasma levels. However, in isolated AMPKα2–/– pancreatic islets, glucose- and L-arginine–stimulated insulin secretion were not affected. AMPKα2–/– mice have reduced insulin-stimulated whole-body glucose utilization and muscle glycogen synthesis rates assessed in vivo by the hyperinsulinemic euglycemic clamp technique. Surprisingly, both parameters were not altered in mice expressing a dominant-negative mutant of AMPK in skeletal muscle. Furthermore, glucose transport was normal in incubated isolated AMPKα2–/– muscles. These data indicate that AMPKα2 in tissues other than skeletal muscles regulates insulin action. Concordantly, we found an increased daily urinary catecholamine excretion in AMPKα2–/– mice, suggesting altered function of the autonomic nervous system that could explain both the impaired insulin secretion and insulin sensitivity observed in vivo. Therefore, extramuscular AMPKα2 catalytic subunit is important for whole-body insulin action in vivo, probably through modulation of sympathetic nervous activity. PMID:12511592

  9. The AMP-activated protein kinase alpha2 catalytic subunit controls whole-body insulin sensitivity.

    PubMed

    Viollet, Benoit; Andreelli, Fabrizio; Jørgensen, Sebastian B; Perrin, Christophe; Geloen, Alain; Flamez, Daisy; Mu, James; Lenzner, Claudia; Baud, Olivier; Bennoun, Myriam; Gomas, Emmanuel; Nicolas, Gaël; Wojtaszewski, Jørgen F P; Kahn, Axel; Carling, David; Schuit, Frans C; Birnbaum, Morris J; Richter, Erik A; Burcelin, Rémy; Vaulont, Sophie

    2003-01-01

    AMP-activated protein kinase (AMPK) is viewed as a fuel sensor for glucose and lipid metabolism. To better understand the physiological role of AMPK, we generated a knockout mouse model in which the AMPKalpha2 catalytic subunit gene was inactivated. AMPKalpha2(-/-) mice presented high glucose levels in the fed period and during an oral glucose challenge associated with low insulin plasma levels. However, in isolated AMPKalpha2(-/-) pancreatic islets, glucose- and L-arginine-stimulated insulin secretion were not affected. AMPKalpha2(-/-) mice have reduced insulin-stimulated whole-body glucose utilization and muscle glycogen synthesis rates assessed in vivo by the hyperinsulinemic euglycemic clamp technique. Surprisingly, both parameters were not altered in mice expressing a dominant-negative mutant of AMPK in skeletal muscle. Furthermore, glucose transport was normal in incubated isolated AMPKalpha2(-/-) muscles. These data indicate that AMPKalpha2 in tissues other than skeletal muscles regulates insulin action. Concordantly, we found an increased daily urinary catecholamine excretion in AMPKalpha2(-/-) mice, suggesting altered function of the autonomic nervous system that could explain both the impaired insulin secretion and insulin sensitivity observed in vivo. Therefore, extramuscular AMPKalpha2 catalytic subunit is important for whole-body insulin action in vivo, probably through modulation of sympathetic nervous activity.

  10. Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.

    PubMed Central

    Biondi, R M; Baehler, P J; Reymond, C D; Véron, M

    1998-01-01

    The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit. PMID:9776758

  11. Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.

    PubMed

    Biondi, R M; Baehler, P J; Reymond, C D; Véron, M

    1998-11-01

    The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit.

  12. Cloning and purification of protein kinase CK2 recombinant alpha and beta subunits from the Mediterranean fly Ceratitis capitata.

    PubMed

    Kouyanou-Koutsoukou, Sophia; Baier, Andrea; Kolaitis, Regina-Maria; Maniatopoulou, Evanthia; Thanopoulou, Konstantina; Szyszka, Ryszard

    2011-10-01

    The Mediterranean fruit fly Ceratitis capitata is an insect capable of wreaking extensive damage to a wide range of fruit crops. Protein kinase CK2 is a ubiquitous Ser/Thr kinase that is highly conserved among eukaryotes; it is a heterotetramer composed of two catalytic (α) and a dimer of regulatory (β) subunits. We present here the construction of the cDNA molecules of the CK2α and CK2β subunits from the medfly C. capitata by the 5'/3' RACE and RT-PCR methods, respectively. CcCK2α catalytic subunit presents the characteristic and conserved features of a typical protein kinase, similar to the regulatory CcCK2β subunit, that also possess the conserved features of regulatory CK2β subunits, as revealed by comparison of their predicted amino acid sequences with other eukaryotic species. The recombinant CcCK2α and CcCK2β proteins were purified by affinity chromatography to homogeneity, after overexpression in Escherichia coli. CcCK2α is capable to utilize GTP and its activity and is inhibited by polyanions and stimulated by polycations in phosphorylation assays, using purified acidic ribosomal protein P1 as a substrate.

  13. A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels

    PubMed Central

    Gradogna, Antonella; Babini, Elena; Picollo, Alessandra

    2010-01-01

    The two human CLC Cl− channels, ClC-Ka and ClC-Kb, are almost exclusively expressed in kidney and inner ear epithelia. Mutations in the genes coding for ClC-Kb and barttin, an essential CLC-K channel β subunit, lead to Bartter syndrome. We performed a biophysical analysis of the modulatory effect of extracellular Ca2+ and H+ on ClC-Ka and ClC-Kb in Xenopus oocytes. Currents increased with increasing [Ca2+]ext without full saturation up to 50 mM. However, in the absence of Ca2+, ClC-Ka currents were still 20% of currents in 10 mM [Ca2+]ext, demonstrating that Ca2+ is not strictly essential for opening. Vice versa, ClC-Ka and ClC-Kb were blocked by increasing [H+]ext with a practically complete block at pH 6. Ca2+ and H+ act as gating modifiers without changing the single-channel conductance. Dose–response analysis suggested that two protons are necessary to induce block with an apparent pK of ∼7.1. A simple four-state allosteric model described the modulation by Ca2+ assuming a 13-fold higher Ca2+ affinity of the open state compared with the closed state. The quantitative analysis suggested separate binding sites for Ca2+ and H+. A mutagenic screen of a large number of extracellularly accessible amino acids identified a pair of acidic residues (E261 and D278 on the loop connecting helices I and J), which are close to each other but positioned on different subunits of the channel, as a likely candidate for forming an intersubunit Ca2+-binding site. Single mutants E261Q and D278N greatly diminished and the double mutant E261Q/D278N completely abolished modulation by Ca2+. Several mutations of a histidine residue (H497) that is homologous to a histidine that is responsible for H+ block in ClC-2 did not yield functional channels. However, the triple mutant E261Q/D278N/H497M completely eliminated H+ -induced current block. We have thus identified a protein region that is involved in binding these physiologically important ligands and that is likely undergoing

  14. Translation and Assembly of Radiolabeled Mitochondrial DNA-Encoded Protein Subunits from Cultured Cells and Isolated Mitochondria.

    PubMed

    Formosa, Luke E; Hofer, Annette; Tischner, Christin; Wenz, Tina; Ryan, Michael T

    2016-01-01

    In higher eukaryotes, the mitochondrial electron transport chain consists of five multi-subunit membrane complexes responsible for the generation of cellular ATP. Of these, four complexes are under dual genetic control as they contain subunits encoded by both the mitochondrial and nuclear genomes, thereby adding another layer of complexity to the puzzle of respiratory complex biogenesis. These subunits must be synthesized and assembled in a coordinated manner in order to ensure correct biogenesis of different respiratory complexes. Here, we describe techniques to (1) specifically radiolabel proteins encoded by mtDNA to monitor the rate of synthesis using pulse labeling methods, and (2) analyze the stability, assembly, and turnover of subunits using pulse-chase methods in cultured cells and isolated mitochondria.

  15. Autocatalytic tyrosine-phosphorylation of protein kinase CK2 alpha and alpha' subunits: implication of Tyr182.

    PubMed Central

    Donella-Deana, A; Cesaro, L; Sarno, S; Brunati, A M; Ruzzene, M; Pinna, L A

    2001-01-01

    CK2 is a pleiotropic and constitutively active serine/threonine protein kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits, whose mechanism of modulation is still obscure. Here we show that CK2 alpha/alpha' subunits undergo intermolecular (trans) tyrosine-autophosphorylation, which is dependent on intrinsic catalytic activity and is suppressed by the individual mutation of Tyr182, a crucial residue of the activation loop, to phenylalanine. At variance with serine-autophosphorylation, tyrosine-autophosphorylation of CK2alpha is reversed by ADP and GDP and is counteracted by the beta-subunit and by a peptide reproducing the activation loop of CK2alpha/alpha' (amino acids 175-201). These results disclose new perspectives about the mode of regulation of CK2 catalytic subunits. PMID:11439109

  16. Regulation of expression of a soybean storage protein subunit gene. Progress report

    SciTech Connect

    Thompson, J.F.; Madison, J.T.

    1984-04-23

    We have found that the methionine repression of the ..beta..-subunit gene expression is not due to degradation of the ..beta..-subunit but is due to an effect on synthesis of the ..beta..-subunit. The effect of methionine on the synthesis of the ..beta..-is due to an inhibition of ..beta..-subunit mRNA synthesis. 3 references, 1 figure.

  17. A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

    PubMed

    Rodermel, S; Haley, J; Jiang, C Z; Tsai, C H; Bogorad, L

    1996-04-30

    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.

  18. Brain Gαi2-subunit proteins and the prevention of salt sensitive hypertension

    PubMed Central

    Carmichael, Casey Y.; Wainford, Richard D.

    2015-01-01

    To counter the development of salt-sensitive hypertension, multiple brain G-protein-coupled receptor (GPCR) systems are activated to facilitate sympathoinhibition, sodium homeostasis, and normotension. Currently there is a paucity of knowledge regarding the role of down-stream GPCR-activated Gα-subunit proteins in these critically important physiological regulatory responses required for long-term blood pressure regulation. We have determined that brain Gαi2-proteins mediate natriuretic and sympathoinhibitory responses produced by acute pharmacological (exogenous central nociceptin/orphanin FQ receptor (NOP) and α2-adrenoceptor activation) and physiological challenges to sodium homeostasis (intravenous volume expansion and 1 M sodium load) in conscious Sprague–Dawley rats. We have demonstrated that in salt-resistant rat phenotypes, high dietary salt intake evokes site-specific up-regulation of hypothalamic paraventricular nucleus (PVN) Gαi2-proteins. Further, we established that PVN Gαi2 protein up-regulation prevents the development of renal nerve-dependent sympathetically mediated salt-sensitive hypertension in Sprague–Dawley and Dahl salt-resistant rats. Additionally, failure to up-regulate PVN Gαi2 proteins during high salt-intake contributes to the pathophysiology of Dahl salt-sensitive (DSS) hypertension. Collectively, our data demonstrate that brain, and likely PVN specific, Gαi2 protein pathways represent a central molecular pathway mediating sympathoinhibitory renal-nerve dependent responses evoked to maintain sodium homeostasis and a salt-resistant phenotype. Further, impairment of this endogenous “anti-hypertensive” mechanism contributes to the pathophysiology of salt-sensitive hypertension. PMID:26347659

  19. The 2.4-A crystal structure of Scapharca dimeric hemoglobin. Cooperativity based on directly communicating hemes at a novel subunit interface.

    PubMed

    Royer, W E; Hendrickson, W A; Chiancone, E

    1989-12-15

    The crystal structure of the cooperative dimeric hemoglobin from the arcid clam, Scapharca inaequivalvis, has been determined in the carbonmonoxy state. The phase problem was solved for reflections with Bragg spacings greater than 3 A using anomalous scattering from the porphyrin iron atoms measured at a single wavelength in combination with molecular averaging. The model built into this electron density map has been refined at 2.4 A resolution by means of stereochemically restrained least squares minimization to a conventional R-value of 0.156. The root mean square deviation from ideal bond lengths and angles are 0.013 A and 1.7 degrees, respectively. In addition to the 2336 hemoglobin atoms, 214 water molecules have been incorporated into the model. This structure reveals the details of an assemblage of two identical myoglobin-like subunits that is radically different from vertebrate hemoglobins. The subunit interface is formed by direct apposition of the E and F helices, whereas these surfaces are external in vertebrate hemoglobins. The interface has both hydrophobic and hydrophilic character. Two symmetrically related hydrophobic regions are formed between subunits. Six residues are involved in each of these regions that pack tightly enough to exclude water but have only a few atoms in close van der Waals contact. A number of ordered water molecules line the interface and form bridging hydrogen bonds between subunits. Four intersubunit ionic interactions are formed, two of which involve negatively charged propionate groups of the porphyrin. In contrast to cooperative vertebrate hemoglobins, a hydrogen bond network provides a direct route for communication between the two heme groups.

  20. Lipases at interfaces: unique interfacial properties as globular proteins.

    PubMed

    Reis, P; Miller, R; Krägel, J; Leser, M; Fainerman, V B; Watzke, H; Holmberg, K

    2008-06-01

    The adsorption behavior of two globular proteins, lipase from Rhizomucor miehei and beta-lactoglobulin, at inert oil/water and air/water interfaces was studied by the pendant drop technique. The kinetics and adsorption isotherms were interpreted for both proteins in different environments. It was found that the adopted mathematical models well describe the adsorption behavior of the proteins at the studied interfaces. One of the main findings is that unique interfacial properties were observed for lipase as compared to the reference beta-lactoglobulin. A folded drop with a "skinlike" film was formed for the two proteins after aging followed by compression. This behavior is normally associated with protein unfolding and covalent cross-linking at the interface. Despite this, the lipase activity was not suppressed. By highlighting the unique interfacial properties of lipases, we believe that the presented work contributes to a better understanding of lipase interfacial activation and the mechanisms regulating lipolysis. The results indicate that the understanding of the physical properties of lipases can lead to novel approaches to regulate their activity.

  1. Cyclic AMP and AKAP-mediated targeting of protein kinase A regulates lactate dehydrogenase subunit A mRNA stability.

    PubMed

    Jungmann, Richard A; Kiryukhina, Olga

    2005-07-01

    Expression of the lactate dehydrogenase A subunit (ldh-A) gene is controlled through transcriptional as well as post-transcriptional mechanisms. Both mechanisms involve activation of protein kinase A (PKA) into its subunits and subsequent phosphorylation and activation of several key regulatory factors. In rat C6 glioma cells, post-transcriptional gene regulation occurs through PKA-mediated stabilization of LDH-A mRNA and subsequent increase of intracellular LDH-A mRNA levels. Previous studies have demonstrated a cAMP-stabilizing region (CSR) located in the LDH-A 3'-untranslated region which, in combination with several phosphorylated CSR-binding proteins (CSR-BP), regulates the PKA-mediated stabilization of LDH-A mRNA. However, the mechanistic details of interaction of CSR with proteins as they pertain to mRNA stabilization by PKA are so far largely unknown. In this study we tested the hypothesis that ribosomal protein extracts (RSW) from glioma cells contain PKA regulatory (RII) and catalytic (C) subunits that, in combination with a protein kinase A anchoring protein (AKAP 95) and CSR-BPs participate in forming CSR-protein complexes that are responsible for mRNA stability regulation. To demonstrate the importance of CSR-protein complex formation, the PKA subunits and AKAP 95 were removed from the RSW by immunoprecipitation, and the antigen-deleted RSW were subjected to CSR binding analysis using gel mobility shift and UV cross-linking. It was shown that AKAP 95 as well as RII formed a direct linkage with CSR during CSR-protein complex formation. In contrast, the catalytic subunit formed part of the CSR-protein complex but did not bind to CSR directly in a covalent linkage. To determine whether formation of CSR complexes that included C, RII, and AKAP 95 constituted a functional event and was necessary for mRNA stabilization, cell-free decay reactions were carried out with RSW extracts, and the kinetics of decay of LDH-A mRNA was determined. Depletion of PKA

  2. Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR

    PubMed Central

    Lee, Sang-Won; Berger, Scott J.; Martinović, Suzana; Paša-Tolić, Ljiljana; Anderson, Gordon A.; Shen, Yufeng; Zhao, Rui; Smith, Richard D.

    2002-01-01

    Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry coupled with capillary reverse-phase liquid chromatography was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accuracy, achieved by “mass locking” with an internal standard from a dual electrospray ionization source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on cotranslational and posttranslational modifications of the ribosomal proteins that included loss of the initiating methionine, acetylation, methylation, and proteolytic maturation. High-resolution separations permitted differentiation of protein isoforms having high structural similarity as well as proteins from their modified forms, facilitating unequivocal assignments. The study identified 42 of the 43 core large ribosomal subunit proteins and 58 (of 64 possible) core large subunit protein isoforms having unique masses in a single analysis. These results demonstrate the basis for the high-throughput analyses of complex mixtures of intact proteins, which we believe will be an important complement to other approaches for defining protein modifications and their changes resulting from physiological processes or environmental perturbations. PMID:11983894

  3. The helical domain of a G protein alpha subunit is a regulator of its effector.

    PubMed

    Liu, W; Northup, J K

    1998-10-27

    The alpha subunit (Galpha) of heterotrimeric G proteins is a major determinant of signaling selectivity. The Galpha structure essentially comprises a GTPase "Ras-like" domain (RasD) and a unique alpha-helical domain (HD). We used the vertebrate phototransduction model to test for potential functions of HD and found that the HD of the retinal transducin Galpha (Galphat) and the closely related gustducin (Galphag), but not Galphai1, Galphas, or Galphaq synergistically enhance guanosine 5'-gamma[-thio]triphosphate bound Galphat (GalphatGTPgammaS) activation of bovine rod cGMP phosphodiesterase (PDE). In addition, both HDt and HDg, but not HDi1, HDs, or HDq attenuate the trypsin-activated PDE. GalphatGDP and HDt attenuation of trypsin-activated PDE saturate with similar affinities and to an identical 38% of initial activity. These data suggest that interaction of intact Galphat with the PDE catalytic core may be caused by the HD moiety, and they indicate an independent site(s) for the HD moiety of Galphat within the PDE catalytic core in addition to the sites for the inhibitory Pgamma subunits. The HD moiety of GalphatGDP is an attenuator of the activated catalytic core, whereas in the presence of activated GalphatGTPgammaS the independently expressed HDt is a potent synergist. Rhodopsin catalysis of Galphat activation enhances the PDE activation produced by subsaturating levels of Galphat, suggesting a HD-moiety synergism from a transient conformation of Galphat. These results establish HD-selective regulations of vertebrate retinal PDE, and they provide evidence demonstrating that the HD is a modulatory domain. We suggest that the HD works in concert with the RasD, enhancing the efficiency of G protein signaling.

  4. Type IV fimbrial subunit protein ApfA contributes to protection against porcine pleuropneumonia

    PubMed Central

    2012-01-01

    Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae accounts for serious economic losses in the pig farming industry worldwide. We examined here the immunogenicity and protective efficacy of the recombinant type IV fimbrial subunit protein ApfA as a single antigen vaccine against pleuropneumonia, or as a component of a multi-antigen preparation comprising five other recombinant antigens derived from key virulence factors of A. pleuropneumoniae (ApxIA, ApxIIA, ApxIIIA, ApxIVA and TbpB). Immunization of pigs with recombinant ApfA alone induced high levels of specific serum antibodies and provided partial protection against challenge with the heterologous A. pleuropneumoniae serotype 9 strain. This protection was higher than that engendered by vaccination with rApxIVA or rTbpB alone and similar to that observed after immunization with the tri-antigen combination of rApxIA, rApxIIA and rApxIIIA. In addition, rApfA improved the vaccination potential of the penta-antigen mixture of rApxIA, rApxIIA, rApxIIIA, rApxIVA and rTbpB proteins, where the hexa-antigen vaccine containing rApfA conferred a high level of protection on pigs against the disease. Moreover, when rApfA was used for vaccination alone or in combination with other antigens, such immunization reduced the number of pigs colonized with the challenge strain. These results indicate that ApfA could be a valuable component of an efficient subunit vaccine for the prevention of porcine pleuropneumonia. PMID:22240397

  5. Role of individual subunits of the Neurospora crassa CSN complex in regulation of deneddylation and stability of cullin proteins.

    PubMed

    Wang, Jiyong; Hu, Qiwen; Chen, Huijie; Zhou, Zhipeng; Li, Weihua; Wang, Ying; Li, Shaojie; He, Qun

    2010-12-02

    The Cop9 signalosome (CSN) is an evolutionarily conserved multifunctional complex that controls ubiquitin-dependent protein degradation in eukaryotes. We found seven CSN subunits in Neurospora crassa in a previous study, but only one subunit, CSN-2, was functionally characterized. In this study, we created knockout mutants for the remaining individual CSN subunits in N. crassa. By phenotypic observation, we found that loss of CSN-1, CSN-2, CSN-4, CSN-5, CSN-6, or CSN-7 resulted in severe defects in growth, conidiation, and circadian rhythm; the defect severity was gene-dependent. Unexpectedly, CSN-3 knockout mutants displayed the same phenotype as wild-type N. crassa. Consistent with these phenotypic observations, deneddylation of cullin proteins in csn-1, csn-2, csn-4, csn-5, csn-6, or csn-7 mutants was dramatically impaired, while deletion of csn-3 did not cause any alteration in the neddylation/deneddylation state of cullins. We further demonstrated that CSN-1, CSN-2, CSN-4, CSN-5, CSN-6, and CSN-7, but not CSN-3, were essential for maintaining the stability of Cul1 in SCF complexes and Cul3 and BTB proteins in Cul3-BTB E3s, while five of the CSN subunits, but not CSN-3 and CSN-5, were also required for maintaining the stability of SKP-1 in SCF complexes. All seven CSN subunits were necessary for maintaining the stability of Cul4-DDB1 complexes. In addition, CSN-3 was also required for maintaining the stability of the CSN-2 subunit and FWD-1 in the SCF(FWD-1) complex. Together, these results not only provide functional insights into the different roles of individual subunits in the CSN complex, but also establish a functional framework for understanding the multiple functions of the CSN complex in biological processes.

  6. Identification of a regulatory subunit of protein phosphatase 1 which mediates blue light signaling for stomatal opening.

    PubMed

    Takemiya, Atsushi; Yamauchi, Shota; Yano, Takayuki; Ariyoshi, Chie; Shimazaki, Ken-ichiro

    2013-01-01

    Protein phosphatase 1 (PP1) is a eukaryotic serine/threonine protein phosphatase comprised of a catalytic subunit (PP1c) and a regulatory subunit that modulates catalytic activity, subcellular localization and substrate specificity. PP1c positively regulates stomatal opening through blue light signaling between phototropins and the plasma membrane H(+)-ATPase in guard cells. However, the regulatory subunit functioning in this process is unknown. We identified Arabidopsis PRSL1 (PP1 regulatory subunit2-like protein1) as a regulatory subunit of PP1c. Tautomycin, a selective inhibitor of PP1c, inhibited blue light responses of stomata in the single mutants phot1 and phot2, supporting the idea that signals from phot1 and phot2 converge on PP1c. We obtained PRSL1 based on the sequence similarity to Vicia faba PRS2, a PP1c-binding protein isolated by a yeast two-hybrid screen. PRSL1 bound to Arabidopsis PP1c through its RVxF motif, a consensus PP1c-binding sequence. Arabidopsis prsl1 mutants were impaired in blue light-dependent stomatal opening, H(+) pumping and phosphorylation of the H(+)-ATPase, but showed normal phototropin activities. PRSL1 complemented the prsl1 phenotype, but not if the protein carried a mutation in the RVxF motif, suggesting that PRSL1 functions through binding PP1c via the RVxF motif. PRSL1 did not affect the catalytic activity of Arabidopsis PP1c but it stimulated the localization of PP1c in the cytoplasm. We conclude that PRSL1 functions as a regulatory subunit of PP1 and regulates blue light signaling in stomata.

  7. Sequential domain assembly of ribosomal protein S3 drives 40S subunit maturation

    PubMed Central

    Mitterer, Valentin; Murat, Guillaume; Réty, Stéphane; Blaud, Magali; Delbos, Lila; Stanborough, Tamsyn; Bergler, Helmut; Leulliot, Nicolas; Kressler, Dieter; Pertschy, Brigitte

    2016-01-01

    Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. We show that S3 dimerizes and is imported into the nucleus with its N-domain in a rotated conformation and associated with the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C-domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins. PMID:26831757

  8. Replication protein A subunit 3 and the iron efficiency response in soybean.

    PubMed

    Atwood, Sarah E; O'Rourke, Jamie A; Peiffer, Gregory A; Yin, Tengfei; Majumder, Mahbubul; Zhang, Chunquan; Cianzio, Silvia R; Hill, John H; Cook, Dianne; Whitham, Steven A; Shoemaker, Randy C; Graham, Michelle A

    2014-01-01

    In soybean [Glycine max (L.) Merr.], iron deficiency results in interveinal chlorosis and decreased photosynthetic capacity, leading to stunting and yield loss. In this study, gene expression analyses investigated the role of soybean replication protein A (RPA) subunits during iron stress. Nine RPA homologs were significantly differentially expressed in response to iron stress in the near isogenic lines (NILs) Clark (iron efficient) and Isoclark (iron inefficient). RPA homologs exhibited opposing expression patterns in the two NILs, with RPA expression significantly repressed during iron deficiency in Clark but induced in Isoclark. We used virus induced gene silencing (VIGS) to repress GmRPA3 expression in the iron inefficient line Isoclark and mirror expression in Clark. GmRPA3-silenced plants had improved IDC symptoms and chlorophyll content under iron deficient conditions and also displayed stunted growth regardless of iron availability. RNA-Seq comparing gene expression between GmRPA3-silenced and empty vector plants revealed massive transcriptional reprogramming with differential expression of genes associated with defense, immunity, aging, death, protein modification, protein synthesis, photosynthesis and iron uptake and transport genes. Our findings suggest the iron efficient genotype Clark is able to induce energy controlling pathways, possibly regulated by SnRK1/TOR, to promote nutrient recycling and stress responses in iron deficient conditions.

  9. The role of Arabidopsis G-protein subunits in MLO2 function and MAMP-triggered immunity

    PubMed Central

    Lorek, Justine; Griebel, Thomas; Jones, Alan M.; Panstruga, Ralph

    2016-01-01

    Heterotrimeric G-proteins are composed of Gα, Gβ and Gγ subunits and regulate many fundamental processes in plants. Nonetheless plants have a considerably simpler repertoire of G-protein signaling components than metazoans. In animals, ligand binding to 7 transmembrane (7TM) cell surface receptors designated GPCRs leads to G protein activation, however, activation of the plant G protein complex is constitutive, therefore the exact role of plant 7TM proteins is unclear. MLOs are the best characterized 7TM plant proteins. While genetic ablation of either MLO2 or G proteins alters resistance to pathogens, it is unknown if G proteins directly couple signaling through MLO2. Here we exploited two well-documented phenotypes of Arabidopsis mlo2 mutants, broad-spectrum powdery mildew resistance and spontaneous callose deposition, to assess the relationship of MLO2 proteins to the G protein complex. Although our data reveal modulation of antifungal defence responses by Gβ and Gγ subunits, our findings are overall inconsistent with a role of MLO2 as a canonical GPCR. We discovered that mutants defective in the Gβ subunit show delayed accumulation of a subset of defence-associated genes following exposure to the microbe-associated molecular pattern (MAMP), flg22. Moreover Gβ mutants were found to be hypersusceptible to spray-inoculation with the bacterial pathogen, Pseudomonas syringae. In sum, our data do not support a function for MLO proteins as GPCRs, but unravel a role for Gβ and Gγ subunits as modulators of basal defence against biotrophic and hemibiotrophic phytopathogens. PMID:23656333

  10. Altered Dimer Interface Decreases Stability in an Amyloidogenic Protein

    SciTech Connect

    Baden, Elizabeth M.; Owen, Barbara A.L.; Peterson, Francis C.; Volkman, Brian F.; Ramirez-Alvarado, Marina; Thompson, James R.

    2008-07-21

    Amyloidoses are devastating and currently incurable diseases in which the process of amyloid formation causes fatal cellular and organ damage. The molecular mechanisms underlying amyloidoses are not well known. In this study, we address the structural basis of immunoglobulin light chain amyloidosis, which results from deposition of light chains produced by clonal plasma cells. We compare light chain amyloidosis protein AL-09 to its wild-type counterpart, the kl O18/O8 light chain germline. Crystallographic studies indicate that both proteins form dimers. However, AL-09 has an altered dimer interface that is rotated 90 degrees from the kl O18/O8 dimer interface. The three non-conservative mutations in AL-09 are located within the dimer interface, consistent with their role in the decreased stability of this amyloidogenic protein. Moreover, AL-09 forms amyloid fibrils more quickly than kl O18/O8 in vitro. These results support the notion that the increased stability of the monomer and delayed fibril formation, together with a properly formed dimer, may be protective against amyloidogenesis. This could open a new direction into rational drug design for amyloidogenic proteins.

  11. Nicotine enhances the cyclic AMP-dependent protein kinase-mediated phosphorylation of alpha4 subunits of neuronal nicotinic receptors.

    PubMed

    Hsu, Y N; Edwards, S C; Wecker, L

    1997-12-01

    Studies determined whether alpha4beta2 or alpha3beta2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP-dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 nM for alpha4beta2 and 500 nM for alpha3beta2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing alpha4beta2 receptors were incubated with [gamma-32P]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the alpha4 subunit was present. Phosphorylation of alpha4 subunits of alpha4beta2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. In contrast, receptors isolated from oocytes expressing alpha3beta2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the alpha3 subunit. Results suggest that the PKA-mediated phosphorylation of alpha4 and not alpha3 subunits may explain the differential inactivation by nicotine of these receptor subtypes expressed in oocytes.

  12. Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin

    PubMed Central

    Fuchs, Julian E.; Huber, Roland G.; Waldner, Birgit J.; Kahler, Ursula; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R.

    2015-01-01

    Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm “dynamics govern specificity” might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design. PMID:26496636

  13. Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin.

    PubMed

    Fuchs, Julian E; Huber, Roland G; Waldner, Birgit J; Kahler, Ursula; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R

    2015-01-01

    Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm "dynamics govern specificity" might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design.

  14. Subunits of highly Fluorescent Protein R-Phycoerythrin as Probes for Cell Imaging and Single-Molecule Detection

    SciTech Connect

    Isailovic, Dragan

    2005-01-01

    The purposes of our research were: (1) To characterize subunits of highly fluorescent protein R-Phycoerythrin (R-PE) and check their suitability for single-molecule detection (SMD) and cell imaging, (2) To extend the use of R-PE subunits through design of similar proteins that will be used as probes for microscopy and spectral imaging in a single cell, and (3) To demonstrate a high-throughput spectral imaging method that will rival spectral flow cytometry in the analysis of individual cells. We first demonstrated that R-PE subunits have spectroscopic and structural characteristics that make them suitable for SMD. Subunits were isolated from R-PE by high-performance liquid chromatography (HPLC) and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). In addition, R-PE subunits and their enzymatic digests were characterized by several separation and detection methods including HPLC, capillary electrophoresis, sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and HPLC-electrospray ionization mass spectrometry (ESI-MS). Favorable absorption and fluorescence of the R-PE subunits and digest peptides originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores that are covalently attached to cysteine residues. High absorption coefficients and strong fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra in the visible region of electromagnetic spectrum, and relatively low molecular weights make these molecules suitable for use as fluorescence labels of biomolecules and cells. We further designed fluorescent proteins both in vitro and in vivo (in Escherichia coli) based on the highly specific attachment of PEB chromophore to genetically expressed apo-subunits of R-PE. In one example, apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii (P. boldii), and expressed in E. coli. Although expressed apo-subunits formed inclusion

  15. Unintended consequences? Water molecules at biological and crystallographic protein-protein interfaces.

    PubMed

    Ahmed, Mostafa H; Habtemariam, Mesay; Safo, Martin K; Scarsdale, J Neel; Spyrakis, Francesca; Cozzini, Pietro; Mozzarelli, Andrea; Kellogg, Glen E

    2013-12-01

    The importance of protein-protein interactions (PPIs) is becoming increasingly appreciated, as these interactions lie at the core of virtually every biological process. Small molecule modulators that target PPIs are under exploration as new therapies. One of the greatest obstacles faced in crystallographically determining the 3D structures of proteins is coaxing the proteins to form "artificial" PPIs that lead to uniform crystals suitable for X-ray diffraction. This work compares interactions formed naturally, i.e., "biological", with those artificially formed under crystallization conditions or "non-biological". In particular, a detailed analysis of water molecules at the interfaces of high-resolution (≤2.30 Å) X-ray crystal structures of protein-protein complexes, where 140 are biological protein-protein complex structures and 112 include non-biological protein-protein interfaces, was carried out using modeling tools based on the HINT forcefield. Surprisingly few and relatively subtle differences were observed between the two types of interfaces: (i) non-biological interfaces are more polar than biological interfaces, yet there is better organized hydrogen bonding at the latter; (ii) biological associations rely more on water-mediated interactions with backbone atoms compared to non-biological associations; (iii) aromatic/planar residues play a larger role in biological associations with respect to water, and (iv) Lys has a particularly large role at non-biological interfaces. A support vector machines (SVMs) classifier using descriptors from this study was devised that was able to correctly classify 84% of the two interface types.

  16. Evolutionary pressure on the topology of protein interface interaction networks.

    PubMed

    Johnson, Margaret E; Hummer, Gerhard

    2013-10-24

    The densely connected structure of protein-protein interaction (PPI) networks reflects the functional need of proteins to cooperate in cellular processes. However, PPI networks do not adequately capture the competition in protein binding. By contrast, the interface interaction network (IIN) studied here resolves the modular character of protein-protein binding and distinguishes between simultaneous and exclusive interactions that underlie both cooperation and competition. We show that the topology of the IIN is under evolutionary pressure, and we connect topological features of the IIN to specific biological functions. To reveal the forces shaping the network topology, we use a sequence-based computational model of interface binding along with network analysis. We find that the more fragmented structure of IINs, in contrast to the dense PPI networks, arises in large part from the competition between specific and nonspecific binding. The need to minimize nonspecific binding favors specific network motifs, including a minimal number of cliques (i.e., fully connected subgraphs) and many disconnected fragments. Validating the model, we find that these network characteristics are closely mirrored in the IIN of clathrin-mediated endocytosis. Features unexpected on the basis of our motif analysis are found to indicate either exceptional binding selectivity or important regulatory functions.

  17. Disassembly of yeast 80S ribosomes into subunits is a concerted action of ribosome-assisted folding of denatured protein.

    PubMed

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-22

    It has been shown by several groups that ribosome can assist folding of denatured protein in vitro and the process is conserved across the species. Domain V of large ribosomal rRNA which occupies the intersubunit side of the large subunit was identified as the key player responsible for chaperoning the folding process. Thus, it is conceivable that denatured protein needs to access the intersubunit space of the ribosome in order to get folded. In this study, we have investigated the mechanism of release of the protein from the eukaryotic ribosome following reactivation. We have observed significant splitting of yeast 80S ribosome when incubated with the denatured BCAII protein. Energy-free disassembly mechanism functions in low Mg(+2) ion concentration for prokaryotic ribosomes. Eukaryotic ribosomes do not show significant splitting even at low Mg(+2) ion concentration. In this respect, denatured protein-induced disassembly of eukaryotic ribosome without the involvement of any external energy source is intriguing. For prokaryotic ribosomes, it was reported that the denatured protein induces ribosome splitting into subunits in order to access domain V-rRNA. In contrast, our results suggest an alternative mechanism for eukaryotic ribosomal rRNA-mediated protein folding and subsequent separation of the subunits by which release of the activated-protein occurs.

  18. Expression profile of G-protein βγ subunit gene transcripts in the mouse olfactory sensory epithelia

    PubMed Central

    Sathyanesan, Aaron; Feijoo, Adrian A.; Mehta, Saloni T.; Nimarko, Akua F.; Lin, Weihong

    2013-01-01

    Heterotrimeric G-proteins mediate a variety of cellular functions, including signal transduction in sensory neurons of the olfactory system. Whereas the Gα subunits in these neurons are well characterized, the gene transcript expression profile of Gβγ subunits is largely missing. Here we report our comprehensive expression analysis to identify Gβ and Gγ subunit gene transcripts in the mouse main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Our reverse transcriptase PCR (RT-PCR) and realtime qPCR analyses of all known Gβ (β1,2,3,4,5) and Gγ (γ1,2,2t,3,4,5,7,8,10,11,12,13) subunits indicate presence of multiple Gβ and Gγ subunit gene transcripts in the MOE and the VNO at various expression levels. These results are supported by our RNA in situ hybridization (RISH) experiments, which reveal the expression patterns of two Gβ subunits and four Gγ subunits in the MOE as well as one Gβ and four Gγ subunits in the VNO. Using double-probe fluorescence RISH and line intensity scan analysis of the RISH signals of two dominant Gβγ subunits, we show that Gγ13 is expressed in mature olfactory sensory neurons (OSNs), while Gβ1 is present in both mature and immature OSNs. Interestingly, we also found Gβ1 to be the dominant Gβ subunit in the VNO and present throughout the sensory epithelium. In contrast, we found diverse expression of Gγ subunit gene transcripts with Gγ2, Gγ3, and Gγ13 in the Gαi2-expressing neuronal population, while Gγ8 is expressed in both layers. Further, we determined the expression of these Gβγ gene transcripts in three post-natal developmental stages (p0, 7, and 14) and found their cell-type specific expression remains largely unchanged, except the transient expression of Gγ2 in a single basal layer of cells in the MOE during P7 and P14. Taken together, our comprehensive expression analyses reveal cell-type specific gene expression of multiple Gβ and Gγ in sensory neurons of the olfactory system. PMID:23759900

  19. Human CDC45 protein binds to minichromosome maintenance 7 protein and the p70 subunit of DNA polymerase alpha.

    PubMed

    Kukimoto, I; Igaki, H; Kanda, T

    1999-11-01

    Budding yeast CDC45 encodes Cdc45p, an essential protein required to trigger initiation of DNA replication in late G1 phase. We cloned four and one species of the human Cdc45p homolog cDNA, resulting from different splicing patterns, from HeLa cell and human placenta cDNA libraries, respectively. A comparison of the cDNAs and the genomic sequence showed that the longest encoding a 610-amino acid protein was comprised of 20 exons. One species, which lacks exon 7 and contains the shorter of two exons 18, was identical with the previously reported CDC45L cDNA and constituted 24 out of 28 clones from HeLa cells. Splicing was different in HeLa cells and TIG-1 cells, a human diploid cell line. Human CDC45 protein was found to bind directly in vitro to human minichromosome maintenance 7 protein (hMCM7) and to the p70 subunit of DNA polymerase alpha. The data support a thesis that human CDC45 acts as a molecular tether to mediate loading of the DNA polymerase alpha on to the DNA replication complex through binding to hMCM7.

  20. cDNA isolation, characterization, and protein intracellular localization of a katanin-like p60 subunit from Arabidopsis thaliana.

    PubMed

    McClinton, R S; Chandler, J S; Callis, J

    2001-01-01

    Katanin, a heterodimeric protein with ATP-dependent microtubule-severing activity, localizes to the centrosome in animal cells. Widespread occurrence is suspected as several species contain homologs to the katanin p60 subunit. Recently we isolated an Arabidopsis thaliana cDNA with significant identity to the p60 subunit of sea urchin katanin. Like p60, the encoded protein is a member of the AAA superfamily of ATPases, containing the Walker ATP binding consensus and the signature AAA minimal consensus sequences within a single larger AAA/CAD amino acid motif. Phylogenetic analysis placed the encoded protein in the AAA subfamily of cytoskeleton-interactive proteins, where it formed a strongly supported clade with 4 other members identified as katanin p60 subunits. The clone was named AtKSS (Arabidopsis thaliana katanin-like protein small subunit). Western blots, performed using a polyclonal antibody raised against recombinant AtKSS, revealed AtKSS is present in protein extracts of all Arabidopsis organs examined. To evaluate potential interactions between AtKSS and the cytoskeleton, the intracellular localization of AtKSS was correlated with that of tubulin. AtKSS was found in perinuclear regions during interphase, surrounding the spindle poles during mitosis, but was absent from the preprophase band and phragmoplast microtubule arrays. These data support the thesis that AtKSS is an Arabidopsis homolog of the p60 subunit of katanin. Its cell cycle-dependent distribution is consistent with microtubule-severing activity, but additional studies will better define its role.

  1. The GIRK1 subunit potentiates G protein activation of cardiac GIRK1/4 hetero-tetramers

    PubMed Central

    Touhara, Kouki K; Wang, Weiwei; MacKinnon, Roderick

    2016-01-01

    G protein gated inward rectifier potassium (GIRK) channels are gated by direct binding of G protein beta-gamma subunits (Gβγ), signaling lipids, and intracellular Na+. In cardiac pacemaker cells, hetero-tetramer GIRK1/4 channels and homo-tetramer GIRK4 channels play a central role in parasympathetic slowing of heart rate. It is known that the Na+ binding site of the GIRK1 subunit is defective, but the functional difference between GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers remains unclear. Here, using purified proteins and the lipid bilayer system, we characterize Gβγ and Na+ regulation of GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers. We find in GIRK4 homo-tetramers that Na+ binding increases Gβγ affinity and thereby increases the GIRK4 responsiveness to G protein stimulation. GIRK1/4 hetero-tetramers are not activated by Na+, but rather are in a permanent state of high responsiveness to Gβγ, suggesting that the GIRK1 subunit functions like a GIRK4 subunit with Na+ permanently bound. DOI: http://dx.doi.org/10.7554/eLife.15750.001 PMID:27074664

  2. Weak conservation of structural features in the interfaces of homologous transient protein-protein complexes.

    PubMed

    Sudha, Govindarajan; Singh, Prashant; Swapna, Lakshmipuram S; Srinivasan, Narayanaswamy

    2015-11-01

    Residue types at the interface of protein-protein complexes (PPCs) are known to be reasonably well conserved. However, we show, using a dataset of known 3-D structures of homologous transient PPCs, that the 3-D location of interfacial residues and their interaction patterns are only moderately and poorly conserved, respectively. Another surprising observation is that a residue at the interface that is conserved is not necessarily in the interface in the homolog. Such differences in homologous complexes are manifested by substitution of the residues that are spatially proximal to the conserved residue and structural differences at the interfaces as well as differences in spatial orientations of the interacting proteins. Conservation of interface location and the interaction pattern at the core of the interfaces is higher than at the periphery of the interface patch. Extents of variability of various structural features reported here for homologous transient PPCs are higher than the variation in homologous permanent homomers. Our findings suggest that straightforward extrapolation of interfacial nature and inter-residue interaction patterns from template to target could lead to serious errors in the modeled complex structure. Understanding the evolution of interfaces provides insights to improve comparative modeling of PPC structures.

  3. Protein Kinase A Catalytic Subunit Primed for Action: Time-Lapse Crystallography of Michaelis Complex Formation.

    PubMed

    Das, Amit; Gerlits, Oksana; Parks, Jerry M; Langan, Paul; Kovalevsky, Andrey; Heller, William T

    2015-12-01

    The catalytic subunit of the cyclic AMP-dependent protein kinase A (PKAc) catalyzes the transfer of the γ-phosphate of bound Mg2ATP to a serine or threonine residue of a protein substrate. Here, time-lapse X-ray crystallography was used to capture a series of complexes of PKAc with an oligopeptide substrate and unreacted Mg2ATP, including the Michaelis complex, that reveal important geometric rearrangements in and near the active site preceding the phosphoryl transfer reaction. Contrary to the prevailing view, Mg(2+) binds first to the M1 site as a complex with ATP and is followed by Mg(2+) binding to the M2 site. Concurrently, the target serine hydroxyl of the peptide substrate rotates away from the active site toward the bulk solvent, which breaks the hydrogen bond with D166. Lastly, the serine hydroxyl of the substrate rotates back toward D166 to form the Michaelis complex with the active site primed for phosphoryl transfer.

  4. Congenital deficiency of two polypeptide subunits of the iron-protein fragment of mitochondrial complex I.

    PubMed

    Moreadith, R W; Cleeter, M W; Ragan, C I; Batshaw, M L; Lehninger, A L

    1987-02-01

    Recently, we described a patient with severe lactic acidosis due to congenital complex I (NADH-ubiquinone oxidoreductase) deficiency. We now report further enzymatic and immunological characterizations. Both NADH and ferricyanide titrations of complex I activity (measured as NADH-ferricyanide reductase) were distinctly altered in the mitochondria from the patient's tissues. In addition, antisera against complex I immunoprecipitated NADH-ferricyanide reductase from the control but not the patient's mitochondria. However, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of complex I polypeptides demonstrated that the majority of the 25 polypeptides comprising complex I were present in the affected mitochondria. A more detailed analysis using subunit selective antisera against the main polypeptides of the iron-protein fragments of complex I revealed a selective absence of the 75- and 13-kD polypeptides. These findings suggest that the underlying basis for this patient's disease was a congenital deficiency of at least two polypeptides comprising the iron-protein fragment of complex I, which resulted in the inability to correctly assemble a functional enzyme complex.

  5. G-Protein α-Subunit Gsα Is Required for Craniofacial Morphogenesis

    PubMed Central

    Wei, Yanxia; Chen, Min; Weinstein, Lee S.; Hong, Yang; Zhu, Minyan; Li, Hongchang; Li, Huashun

    2016-01-01

    The heterotrimeric G protein subunit Gsα couples receptors to activate adenylyl cyclase and is required for the intracellular cAMP response and protein kinase A (PKA) activation. Gsα is ubiquitously expressed in many cell types; however, the role of Gsα in neural crest cells (NCCs) remains unclear. Here we report that NCCs-specific Gsα knockout mice die within hours after birth and exhibit dramatic craniofacial malformations, including hypoplastic maxilla and mandible, cleft palate and craniofacial skeleton defects. Histological and anatomical analysis reveal that the cleft palate in Gsα knockout mice is a secondary defect resulting from craniofacial skeleton deficiencies. In Gsα knockout mice, the morphologies of NCCs-derived cranial nerves are normal, but the development of dorsal root and sympathetic ganglia are impaired. Furthermore, loss of Gsα in NCCs does not affect cranial NCCs migration or cell proliferation, but significantly accelerate osteochondrogenic differentiation. Taken together, our study suggests that Gsα is required for neural crest cells-derived craniofacial development. PMID:26859889

  6. Environmental reprogramming of the expression of protein kinase CK2beta subunit in fish.

    PubMed

    Alvarez, M; Kausel, G; Figueroa, J; Vera, M I

    2001-11-01

    The dramatic segregation of the nucleolar components in winter-acclimatized carp is the most striking cellular-phenotypical feature observed during the seasonal adaptation of this fish toward the circannual changes in its habitat. Our studies also show that the carp habitat temperature and photoperiod winter conditions provoke a remarkable reduction of both rRNA transcription and the processing of their precursors. To gain knowledge on the mechanisms involved in the regulation of nucleolar activity during the seasonal adaptation process, we studied the behavior of some genes, specifically snoRNA U3 and protein kinase CK2. Consistent with the reduction in the synthesis and processing of pre-rRNA observed during the cold season, the level of CK2beta expression decreases in winter when compared to that attained in summer. Similarly, in winter, liver and kidney cells contain lower levels of CK2beta subunit protein compared to summer. CK2 is associated with or modifies different factors and enzymes involved in the nucleolar activity; therefore, its higher or lower content could be part of the molecular mechanisms underlying the nucleolar seasonal changes that occur during the compensatory acclimatization process.

  7. Dictyostelium discoideum protein phosphatase-1 catalytic subunit exhibits distinct biochemical properties.

    PubMed Central

    Andrioli, Luiz P M; Zaini, Paulo A; Viviani, Wladia; Da Silva, Aline M

    2003-01-01

    Protein phosphatase-1 (PP1) is expressed ubiquitously and is involved in many eukaryotic cellular functions, although PP1 enzyme activity could not be detected in the social amoeba Dictyostelium discoideum cell extracts. In the present paper, we show that D. discoideum has a single copy gene that codes for the catalytic subunit of PP1 (DdPP1c). DdPP1c is expressed throughout the D. discoideum life cycle with constant levels of mRNA, and its protein and amino acid sequence show a mean identity of 80% with other PP1c enzymes. However, it has a distinctive difference: the substitution of a phenylalanine residue (Phe(269) in the DdPP1c) for a highly conserved cysteine residue (Cys(273) in rabbit PP1c) in a region that was shown to have a critical role in the interaction of rabbit PP1c with toxin inhibitors. Wild-type DdPP1c and an engineered mutant form in which Phe(269) was replaced by a cysteine residue were expressed in Escherichia coli. Both recombinant activities were similarly inhibited by okadaic acid, tautomycin and microcystin. However, the Phe(269)-->Cys mutation resulted in a large increase in enzyme activity towards phosphorylase a and a higher sensitivity to calyculin A. These results, together with the molecular modelling of DdPP1c structure, indicate that the Phe(269) residue, which occurs naturally in D. discoideum, confers distinct biochemical properties on this enzyme. PMID:12737629

  8. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Garbers, C.; DeLong, A.; Deruere, J.; Bernasconi, P.; Soll, D.; Evans, M. L. (Principal Investigator)

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

  9. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

    PubMed Central

    Garbers, C; DeLong, A; Deruére, J; Bernasconi, P; Söll, D

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis. Images PMID:8641277

  10. Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus.

    PubMed Central

    Ruediger, R; Roeckel, D; Fait, J; Bergqvist, A; Magnusson, G; Walter, G

    1992-01-01

    Protein phosphatase 2A is composed of three subunits: the catalytic subunit C and two regulatory subunits, A and B. The A subunit consists of 15 nonidentical repeats and has a rodlike shape. It is associated with the B and C subunits as well as with the simian virus 40 small T, polyomavirus small T, and polyomavirus medium T tumor antigens. We determined the binding sites on subunit A for subunit C and tumor antigens by site-directed mutagenesis of A. Twenty-four N- and C-terminal truncations and internal deletions of A were assayed by coimmunoprecipitation for their ability to bind C and tumor antigens. It was found that C binds to repeats 11 to 15 at the C terminus of A, whereas T antigens bind to overlapping but distinct regions of the N terminus. Simian virus 40 small T binds to repeats 3 to 6, and polyomavirus small T and medium T bind to repeats 2 to 8. The data suggest cooperativity between C and T antigens in binding to A. This is most apparent for medium T antigen, which can only bind to those A subunit molecules that provide the entire binding region for the C subunit. We infer from our results that B also binds to N-terminal repeats. A model of the small T/medium T/B-A-C complexes is presented. Images PMID:1328865

  11. Analysis of the interface variability in NMR structure ensembles of protein-protein complexes.

    PubMed

    Calvanese, Luisa; D'Auria, Gabriella; Vangone, Anna; Falcigno, Lucia; Oliva, Romina

    2016-06-01

    NMR structures consist in ensembles of conformers, all satisfying the experimental restraints, which exhibit a certain degree of structural variability. We analyzed here the interface in NMR ensembles of protein-protein heterodimeric complexes and found it to span a wide range of different conservations. The different exhibited conservations do not simply correlate with the size of the systems/interfaces, and are most probably the result of an interplay between different factors, including the quality of experimental data and the intrinsic complex flexibility. In any case, this information is not to be missed when NMR structures of protein-protein complexes are analyzed; especially considering that, as we also show here, the first NMR conformer is usually not the one which best reflects the overall interface. To quantify the interface conservation and to analyze it, we used an approach originally conceived for the analysis and ranking of ensembles of docking models, which has now been extended to directly deal with NMR ensembles. We propose this approach, based on the conservation of the inter-residue contacts at the interface, both for the analysis of the interface in whole ensembles of NMR complexes and for the possible selection of a single conformer as the best representative of the overall interface. In order to make the analyses automatic and fast, we made the protocol available as a web tool at: https://www.molnac.unisa.it/BioTools/consrank/consrank-nmr.html.

  12. Comprehensive inventory of protein complexes in the Protein Data Bank from consistent classification of interfaces

    SciTech Connect

    Bordner, Andrew J.; Gorin, Andrey A.

    2008-05-12

    Here, protein-protein interactions are ubiquitous and essential for cellular processes. High-resolution X-ray crystallographic structures of protein complexes can elucidate the details of their function and provide a basis for many computational and experimental approaches. Here we demonstrate that existing annotations of protein complexes, including those provided by the Protein Data Bank (PDB) itself, contain a significant fraction of incorrect annotations. Results: We have developed a method for identifying protein complexes in the PDB X-ray structures by a four step procedure: (1) comprehensively collecting all protein-protein interfaces; (2) clustering similar protein-protein interfaces together; (3) estimating the probability that each cluster is relevant based on a diverse set of properties; and (4) finally combining these scores for each entry in order to predict the complex structure. Unlike previous annotation methods, consistent prediction of complexes with identical or almost identical protein content is insured. The resulting clusters of biologically relevant interfaces provide a reliable catalog of evolutionary conserved protein-protein interactions.

  13. Comprehensive inventory of protein complexes in the Protein Data Bank from consistent classification of interfaces

    DOE PAGES

    Bordner, Andrew J.; Gorin, Andrey A.

    2008-05-12

    Here, protein-protein interactions are ubiquitous and essential for cellular processes. High-resolution X-ray crystallographic structures of protein complexes can elucidate the details of their function and provide a basis for many computational and experimental approaches. Here we demonstrate that existing annotations of protein complexes, including those provided by the Protein Data Bank (PDB) itself, contain a significant fraction of incorrect annotations. Results: We have developed a method for identifying protein complexes in the PDB X-ray structures by a four step procedure: (1) comprehensively collecting all protein-protein interfaces; (2) clustering similar protein-protein interfaces together; (3) estimating the probability that each cluster ismore » relevant based on a diverse set of properties; and (4) finally combining these scores for each entry in order to predict the complex structure. Unlike previous annotation methods, consistent prediction of complexes with identical or almost identical protein content is insured. The resulting clusters of biologically relevant interfaces provide a reliable catalog of evolutionary conserved protein-protein interactions.« less

  14. Topography and stoichiometry of acidic proteins in large ribosomal subunits from Artemia salina as determined by crosslinking

    SciTech Connect

    Uchiumi, T.; Wahba, A.J.; Traut, R.R.

    1987-08-01

    The 60S subunits isolated from Artemia salina ribosomes were treated with the crosslinking reagent 2-iminothiolane under mild conditions. Proteins were extracted and fractions containing crosslinked acidic proteins were obtained by stepwise elution from CM-cellulose. Each fraction was analyzed by diagonal (two-dimensional nonreducing-reducing) NaDodSO/sub 4//polyacrylamide gel electrophoresis. Crosslinked proteins below the diagonal were radioiodinated and identified by two-dimensional acidic urea-NaDodSO/sub 4/ gel electrophoresis. Each of the acidic proteins P1 and P2 was crosslinked individually to the same third protein, PO. The fractions containing acidic proteins were also analyzed by two-dimensional nonequilibrium isoelectric focusing-NaDodSO/sub 4//polyacrylamide gel electrophoresis. Two crosslinked complexes were observed that coincide in isoelectric positions with monomeric P1 and P2, respectively. Both P1 and P2 appear to form crosslinked homodimers. These results suggest the presence in the 60S subunit of (P1)/sub 2/ and (P2)/sub 2/ dimers, each of which is anchored to PO. Protein PO appears to play the same role as L10 in Escherichia coli ribosomes and may form a pentameric complex with the two dimers in the 60S subunits.

  15. The Cytoplasmic Rhodopsin-Protein Interface: Potential for Drug Discovery

    PubMed Central

    Yanamala, Naveena; Gardner, Eric; Riciutti, Alec; Klein-Seetharaman, Judith

    2011-01-01

    The mammalian dim-light photoreceptor rhodopsin is a prototypic G protein coupled receptor (GPCR), interacting with the G protein, transducin, rhodopsin kinase, and arrestin. All of these proteins interact with rhodopsin at its cytoplasmic surface. Structural and modeling studies have provided in-depth descriptions of the respective interfaces. Overlap and thus competition for binding surfaces is a major regulatory mechanism for signal processing. Recently, it was found that the same surface is also targeted by small molecules. These ligands can directly interfere with the binding and activation of the proteins of the signal transduction cascade, but they can also allosterically modulate the retinal ligand binding pocket. Because the pocket that is targeted contains residues that are highly conserved across Class A GPCRs, these findings imply that it may be possible to target multiple GPCRs with the same ligand(s). This is desirable for example in complex diseases such as cancer where multiple GPCRs participate in the disease networks. PMID:21777183

  16. Conformational dynamics of amyloid proteins at the aqueous interface

    NASA Astrophysics Data System (ADS)

    Armbruster, Matthew; Horst, Nathan; Aoki, Brendy; Malik, Saad; Soto, Patricia

    2013-03-01

    Amyloid proteins is a class of proteins that exhibit distinct monomeric and oligomeric conformational states hallmark of deleterious neurological diseases for which there are not yet cures. Our goal is to examine the extent of which the aqueous/membrane interface modulates the folding energy landscape of amyloid proteins. To this end, we probe the dynamic conformational ensemble of amyloids (monomer prion protein and Alzheimer's Ab protofilaments) interacting with model bilayers. We will present the results of our coarse grain molecular modeling study in terms of the existence of preferential binding spots of the amyloid to the bilayer and the response of the bilayer to the interaction with the amyloid. NSF Nebraska EPSCoR First Award

  17. IChemPIC: A Random Forest Classifier of Biological and Crystallographic Protein-Protein Interfaces.

    PubMed

    Da Silva, Franck; Desaphy, Jérémy; Bret, Guillaume; Rognan, Didier

    2015-09-28

    Protein-protein interactions are becoming a major focus of academic and pharmaceutical research to identify low molecular weight compounds able to modulate oligomeric signaling complexes. As the number of protein complexes of known three-dimensional structure is constantly increasing, there is a need to discard biologically irrelevant interfaces and prioritize those of high value for potential druggability assessment. A Random Forest model has been trained on a set of 300 protein-protein interfaces using 45 molecular interaction descriptors as input. It is able to predict the nature of external test interfaces (crystallographic vs biological) with accuracy at least equal to that of the best state-of-the-art methods. However, our method presents unique advantages in the early prioritization of potentially ligandable protein-protein interfaces: (i) it is equally robust in predicting either crystallographic or biological contacts and (ii) it can be applied to a wide array of oligomeric complexes ranging from small-sized biological interfaces to large crystallographic contacts.

  18. Are protein-protein interfaces special regions on a protein’s surface?

    PubMed Central

    Skolnick, Jeffrey

    2015-01-01

    Protein-protein interactions (PPIs) are involved in many cellular processes. Experimentally obtained protein quaternary structures provide the location of protein-protein interfaces, the surface region of a given protein that interacts with another. These regions are termed half-interfaces (HIs). Canonical HIs cover roughly one third of a protein’s surface and were found to have more hydrophobic residues than the non-interface surface region. In addition, the classical view of protein HIs was that there are a few (if not one) HIs per protein that are structurally and chemically unique. However, on average, a given protein interacts with at least a dozen others. This raises the question of whether they use the same or other HIs. By copying HIs from monomers with the same folds in solved quaternary structures, we introduce the concept of geometric HIs (HIs whose geometry has a significant match to other known interfaces) and show that on average they cover three quarters of a protein’s surface. We then demonstrate that in some cases, these geometric HI could result in real physical interactions (which may or may not be biologically relevant). The composition of the new HIs is on average more charged compared to most known ones, suggesting that the current protein interface database is biased towards more hydrophobic, possibly more obligate, complexes. Finally, our results provide evidence for interface fuzziness and PPI promiscuity. Thus, the classical view of unique, well defined HIs needs to be revisited as HIs are another example of coarse-graining that is used by nature. PMID:26723634

  19. Brain region specific alterations in the protein and mRNA levels of protein kinase A subunits in the post-mortem brain of teenage suicide victims.

    PubMed

    Pandey, Ghanshyam N; Dwivedi, Yogesh; Ren, Xinguo; Rizavi, Hooriyah S; Mondal, Amal C; Shukla, Pradeep K; Conley, Robert R

    2005-08-01

    Protein kinase A (PKA), a critical component of the adenylyl cyclase signaling system, phosphorylates crucial proteins and has been implicated in the pathophysiology of depression and suicide. The objective of the study was to examine if changes in PKA activity or in the protein and messenger RNA (mRNA) expression of any of its subunits are related to the pathophysiology of teenage suicide. We determined PKA activity and the protein and mRNA expression of different subunits of PKA in cytosol and membrane fractions obtained from the prefrontal cortex, (PFC) hippocampus, and nucleus accumbens (NA) of post-mortem brain from 17 teenage suicide victims and 17 nonpsychiatric control subjects. PKA activity was significantly decreased in the PFC but not the hippocampus of teenage suicide victims as compared with controls. However, the protein and mRNA expression of only two PKA subunits, that is, PKA RIalpha and PKA RIbeta, but not any other subunits were significantly decreased in both membrane and cytosol fractions of the PFC and protein expression of RIalpha and RIbeta in the NA of teenage suicide victims as compared to controls. A decrease in protein and mRNA expression of two specific PKA subunits may be associated with the pathogenesis of teenage suicide, and this decrease may be brain region specific, which may be related to the specific behavioral functions associated with these brain areas. Whether these changes in PKA subunits are related to suicidal behavior or are a result of suicide or are specific to suicide is not clear at this point.

  20. Proteins at flowing interfaces: From understanding structure to treating disease

    NASA Astrophysics Data System (ADS)

    Posada, David; Young, James; Hirsa, Amir

    2012-11-01

    The field of soft matter offers vast opportunities for scientific and technological developments, with many challenges that need to be addressed by various disciplines. Fluid dynamics has a tremendous potential for greater impact, from broadening fundamental understanding to treating disease. Here we demonstrate the use of fluid dynamics in two biotechnology problems involving proteins at the air/water interface: a) 2-Dimensional protein crystallization and b) amyloid fibril formation. Protein crystallization is usually the most challenging step in X-ray diffraction analysis of protein structure. Recently it was demonstrated that flow can induce 2-D protein crystallization at conditions under which quiescent systems do not form crystals. A different form of protein structuring, namely amyloid fibrillization, is also of interest due to its association with several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Protein denaturation, which is the root of the fibrillization process, is also a significant concern in biotherapeutics production. Both problems are studied by using shearing free-surface flows in simple geometries. The common finding is that flow can significantly enhance the growth of protein structures.

  1. Giα and Gβ subunits both define selectivity of G protein activation by α2-adrenergic receptors

    PubMed Central

    Gibson, Scott K.; Gilman, Alfred G.

    2006-01-01

    Previous studies of the specificity of receptor interactions with G protein subunits in living cells have relied on measurements of second messengers or other downstream responses. We have examined the selectivity of interactions between α2-adrenergic receptors (α2R) and various combinations of Giα and Gβ subunit isoforms by measuring changes in FRET between Giα–yellow fluorescent protein and cyan fluorescent protein–Gβ chimeras in HeLa cells. All combinations of Giα1, -2, or -3 with Gβ1, -2, or -4 were activated to some degree by endogenous α2Rs as judged by agonist-dependent decreases in FRET. The degree of G protein activation is determined by the combination of Giα and Gβ subunits rather than by the identity of an individual subunit. RT-PCR analysis and small interfering RNA knockdown of α2R subtypes, followed by quantification of radiolabeled antagonist binding, demonstrated that HeLa cells express α2a- and α2b-adrenergic receptor isoforms in a 2:1 ratio. Increasing receptor number by overexpression of the α2aR subtype minimized the differences among coupling preferences for Giα and Gβ isoforms. The molecular properties of each Giα, Gβ, and α2-adrenergic receptor subtype influence signaling efficiency for the α2-adrenergic receptor-mediated signaling pathway. PMID:16371464

  2. Specific Subunits of Heterotrimeric G Proteins Play Important Roles during Nodulation in Soybean1[W][OA

    PubMed Central

    Choudhury, Swarup Roy; Pandey, Sona

    2013-01-01

    Heterotrimeric G proteins comprising Gα, Gβ, and Gγ subunits regulate many fundamental growth and development processes in all eukaryotes. Plants possess a relatively limited number of G-protein components compared with mammalian systems, and their detailed functional characterization has been performed mostly in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). However, the presence of single Gα and Gβ proteins in both these species has significantly undermined the complexity and specificity of response regulation in plant G-protein signaling. There is ample pharmacological evidence for the role of G proteins in regulation of legume-specific processes such as nodulation, but the lack of genetic data from a leguminous species has restricted its direct assessment. Our recent identification and characterization of an elaborate G-protein family in soybean (Glycine max) and the availability of appropriate molecular-genetic resources have allowed us to directly evaluate the role of G-protein subunits during nodulation. We demonstrate that all G-protein genes are expressed in nodules and exhibit significant changes in their expression in response to Bradyrhizobium japonicum infection and in representative supernodulating and nonnodulating soybean mutants. RNA interference suppression and overexpression of specific G-protein components results in lower and higher nodule numbers, respectively, validating their roles as positive regulators of nodule formation. Our data further show preferential usage of distinct G-protein subunits in the presence of an additional signal during nodulation. Interestingly, the Gα proteins directly interact with the soybean nodulation factor receptors NFR1α and NFR1β, suggesting that the plant G proteins may couple with receptors other than the canonical heptahelical receptors common in metazoans to modulate signaling. PMID:23569109

  3. E6-Associated Protein Dependent Estrogen Receptor Regulation of Protein Kinase A Regulatory Subunit R2A Expression in Neuroblastoma.

    PubMed

    Obeid, Jean-Pierre; Zeidan, Youssef H; Zafar, Nawal; El Hokayem, Jimmy

    2017-02-18

    E6ap is a known transcriptional coregulator for estrogen receptor alpha (Er, Erα) in the presence of estrogen. Protein kinase A (PKA) contains two regulatory subunits derived from four genes. Recent evidence demonstrates that PKA regulates E6ap activity. Data generated in our lab indicated estrogen dependent regulation of Pkar2a levels. Our project sets to investigate a possible feedback mechanism constituting of Erα and E6ap transcriptional regulation of Pkar2a expression. Western blot evaluated protein regulation correlations with E2 in mouse neuroblastoma lines. Bioinformatics detected estrogen response element (ERE) sequences. quantitative polymerase chain reaction (qPCR) validated the western blot results. ERE oligonucleotides were synthesized. Reporter gene transcriptional activity was evaluated via Luciferase assay output. Electromobility shift assay (EMSA) assessed direct binding between Erα relevant sequences. Chromatin immunoprecipitation (ChIP) and Re-ChIP were conducted in quantifying protein complex recruitment levels. Pkar2a protein expression directly correlated with E2, and four putative ERE sequences were identified. Pkar2a mRNA expression reverted to baseline with either E2 or E6ap absent. In the presence of E2, ERE-1 and ERE-4 possessed Luciferase reporter gene transcriptional capabilities. ERE-1 portrayed band shifts, representing direct binding to Erα with E2 supplementation. With E2, ERE-1 significantly enhanced Erα and E6ap recruitment levels to the Pkar2a promoter. Pkar2a is directly regulated by Erα and E6ap in the presence of estrogen stimulus. This work indicates a feedback mechanism in the interplay between PKA and E6ap, which may prove crucial for the role of both proteins in cancers and neurogenetic diseases like Angelman syndrome.

  4. Identification of the subunit proteins of 10-nm neurofilaments isolated from axoplasm of squid and Myxicola giant axons

    PubMed Central

    1979-01-01

    Neurofilaments were isolated from the axoplasm of the giant axons of Myxicola infundibulum and squid. The axoplasm was fractionated by discontinuous sucrose gradient centrifugation and gel filtration on Sepharose 4B. The fractions were monitored for neurofilaments by electron microscopy. When isolated in the presence of chelating agents, the neurofilaments of Myxicola are composed almost entirely of protein subunits with mol wt of 150,000 and 160,000. Squid neurofilaments contain two major proteins with mol wt of 200,000 and 60,000. These proteins are compared with other intermediate filament proteins which have been reported in the literature. PMID:479305

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-10-18

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

  7. Fine mapping of sequential neutralization epitopes on the subunit protein VP8 of human rotavirus.

    PubMed Central

    Kovacs-Nolan, Jennifer; Yoo, Dongwan; Mine, Yoshinori

    2003-01-01

    The epitopes of the HRV (human rotavirus), especially those involved in virus neutralization, have not been determined in their entirety, and would have significant implications for HRV vaccine development. In the present study, we report on the epitope mapping and identification of sequential neutralization epitopes, on the Wa strain HRV subunit protein VP8, using synthetic overlapping peptides. Polyclonal antibodies against recombinant Wa VP8 were produced previously in chicken, and purified from egg yolk, which showed neutralizing activity against HRV in vitro. Overlapping VP8 peptide fragments were synthesized and probed with the anti-VP8 antibodies, revealing five sequential epitopes on VP8. Further analysis suggested that three of the five epitopes detected, M1-L10, I55-D66 and L223-P234, were involved in virus neutralization, indicating that sequential epitopes may also be important for the HRV neutralization. The interactions of the antibodies with the five epitopes were characterized by an examination of the critical amino acids involved in antibody binding. Epitopes comprised primarily of hydrophobic amino acid residues, followed by polar and charged residues. The more critical amino acids appeared to be located near the centre of the epitopes, with proline, isoleucine, serine, glutamine and arginine playing an important role in the binding of antibody to the VP8 epitopes. PMID:12901721

  8. Expression of a Ricin Toxin B Subunit: Insulin Fusion Protein in Edible Plant Tissues

    PubMed Central

    Carter, James E.; Odumosu, Oludare; Langridge, William H. R.

    2013-01-01

    Onset of juvenile Type 1 diabetes (T1D) occurs when autoreactive lymphocytes progressively destroy the insulin-producing beta-cells in the pancreatic Islets of Langerhans. The increasing lack of insulin and subsequent onset of hyperglycemia results in increased damage to nerves, blood vessels, and tissues leading to the development of a host of severe disease symptoms resulting in premature morbidity and mortality. To enhance restoration of normoglycemia and immunological homeostasis generated by lymphocytes that mediate the suppression of autoimmunity, the non-toxic B chain of the plant AB enterotoxin ricin (RTB), a castor bean lectin binding a variety of epidermal cell receptors, was genetically linked to the coding region of the proinsulin gene (INS) and expressed as a fusion protein (INS–RTB) in transformed potato plants. This study is the first documented example of a plant enterotoxin B subunit linked to an autoantigen and expressed in transgenic plants for enhanced immunological suppression of T1D autoimmunity. PMID:19898971

  9. G Protein βγ-subunit signaling mediates airway hyperresponsiveness and inflammation in allergic asthma.

    PubMed

    Nino, Gustavo; Hu, Aihua; Grunstein, Judith S; McDonough, Joseph; Kreiger, Portia A; Josephson, Maureen B; Choi, John K; Grunstein, Michael M

    2012-01-01

    Since the Gβγ subunit of Gi protein has been importantly implicated in regulating immune and inflammatory responses, this study investigated the potential role and mechanism of action of Gβγ signaling in regulating the induction of airway hyperresponsiveness (AHR) in a rabbit model of allergic asthma. Relative to non-sensitized animals, OVA-sensitized rabbits challenged with inhaled OVA exhibited AHR, lung inflammation, elevated BAL levels of IL-13, and increased airway phosphodiesterase-4 (PDE4) activity. These proasthmatic responses were suppressed by pretreatment with an inhaled membrane-permeable anti-Gβγ blocking peptide, similar to the suppressive effect of glucocorticoid pretreatment. Extended mechanistic studies demonstrated that: 1) corresponding proasthmatic changes in contractility exhibited in isolated airway smooth muscle (ASM) sensitized with serum from OVA-sensitized+challenged rabbits or IL-13 were also Gβγ-dependent and mediated by MAPK-upregulated PDE4 activity; and 2) the latter was attributed to Gβγ-induced direct stimulation of the non-receptor tyrosine kinase, c-Src, resulting in downstream activation of ERK1/2 and its consequent transcriptional upregulation of PDE4. Collectively, these data are the first to identify that a mechanism involving Gβγ-induced direct activation of c-Src, leading to ERK1/2-mediated upregulation of PDE4 activity, plays a decisive role in regulating the induction of AHR and inflammation in a rabbit model of allergic airway disease.

  10. Phosphoryl transfer reaction snapshots in crystals: Insights into the mechanism of protein kinase a catalytic subunit

    DOE PAGES

    Das, Amit; Gerlits, Oksana O.; Heller, William T.; ...

    2015-06-19

    To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca2+ ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, themore » thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca2+ cations with Mg2+ ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. As a result, the present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date.« less

  11. Phosphoryl transfer reaction snapshots in crystals: Insights into the mechanism of protein kinase a catalytic subunit

    SciTech Connect

    Das, Amit; Gerlits, Oksana O.; Heller, William T.; Kovalevskyi, Andrii Y.; Langan, Paul; Tian, Jianhui

    2015-06-19

    To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca2+ ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, the thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca2+ cations with Mg2+ ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. As a result, the present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date.

  12. Phosphoryl Transfer Reaction Snapshots in Crystals: INSIGHTS INTO THE MECHANISM OF PROTEIN KINASE A CATALYTIC SUBUNIT.

    PubMed

    Gerlits, Oksana; Tian, Jianhui; Das, Amit; Langan, Paul; Heller, William T; Kovalevsky, Andrey

    2015-06-19

    To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca(2+) ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, the thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca(2+) cations with Mg(2+) ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. The present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date.

  13. The use of dissolved oxygen-controlled, fed-batch aerobic cultivation for recombinant protein subunit vaccine manufacturing.

    PubMed

    Farrell, Patrick; Sun, Jacob; Champagne, Paul-Philippe; Lau, Heron; Gao, Meg; Sun, Hong; Zeiser, Arno; D'Amore, Tony

    2015-11-27

    A simple "off-the-shelf" fed-batch approach to aerobic bacterial cultivation for recombinant protein subunit vaccine manufacturing is presented. In this approach, changes in the dissolved oxygen levels are used to adjust the nutrient feed rate (DO-stat), so that the desired dissolved oxygen level is maintained throughout cultivation. This enables high Escherichia coli cell densities and recombinant protein titers. When coupled to a kLa-matched scale-down model, process performance is shown to be consistent at the 2L, 20L, and 200L scales for two recombinant E. coli strains expressing different protein subunit vaccine candidates. Additionally, by mining historical DO-stat nutrient feeding data, a method to transition from DO-stat to a pre-determined feeding profile suitable for larger manufacturing scales without using feedback control is demonstrated at the 2L, 20L, and 200L scales.

  14. Dominant-negative mutants of a yeast G-protein beta subunit identify two functional regions involved in pheromone signalling.

    PubMed Central

    Leberer, E; Dignard, D; Hougan, L; Thomas, D Y; Whiteway, M

    1992-01-01

    The STE4 gene, which encodes the beta subunit of the mating response G-protein in the yeast Saccharomyces cerevisiae, was subjected to a saturation mutagenesis using 'doped' oligodeoxynucleotides. We employed a genetic screen to select dominant-negative STE4 mutants, which when overexpressed from the GAL1 promoter, interfered with the signalling function of the wild type protein. The identified inhibitory amino acid alterations define two small regions that are crucially involved in transmitting the mating signal from G beta to downstream components of the signalling pathway. These results underline the positive signalling role of yeast G beta and assign for the first time the positive signalling function of a G-protein beta subunit to specific structural features. Images PMID:1464310

  15. Glucagon induces disaggregation of polymer-like structures of the. alpha. subunit of the stimulatory G protein in liver membranes

    SciTech Connect

    Nakamura, Shunichi; Rodbell, M. )

    1991-08-15

    The hydrodynamic behavior of G{alpha}{sub s}, the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein (G protein), in octyl glucoside extracts of rat liver membranes was investigated. As was previously shown for G proteins similarly extracted from brain synaptoneurosomes, G{alpha}{sub s} behaved as polydisperse structures with S values higher than that of heterotrimeric G proteins. When G{alpha}{sub s} in its membrane-bound form was ({sup 32}P)ADP-ribosylated by cholera toxin and the treated membranes were extracted with octyl glucoside, > 35% of the labeled G{alpha}{sub s} was found in material that sedimented through sucrose gradients and contained relatively low levels of immunoreactive G{alpha}{sub s}. These finding suggest that the glucagon receptor selectivity interacts with polymer-like structures of G{alpha}{sub 2} and that activation by GTP({gamma}S) results in disaggregation. The role of the {beta} and {gamma} subunits of G proteins in the hormone-induced process is not clear since the polymer-like structures extracted with octyl glucoside are devoid of {beta} and {gamma} subunits.

  16. Evolution, expression differentiation and interaction specificity of heterotrimeric G-protein subunit gene family in the mesohexaploid Brassica rapa.

    PubMed

    Arya, Gulab C; Kumar, Roshan; Bisht, Naveen C

    2014-01-01

    Heterotrimeric G-proteins, comprising of Gα, Gβ, and Gγ subunits, are important signal transducers which regulate many aspects of fundamental growth and developmental processes in all eukaryotes. Initial studies in model plants Arabidopsis and rice suggest that the repertoire of plant G-protein is much simpler than that observed in metazoans. In order to assess the consequence of whole genome triplication events within Brassicaceae family, we investigated the multiplicity of G-protein subunit genes in mesohexaploid Brassica rapa, a globally important vegetable and oilseed crop. We identified one Gα (BraA.Gα1), three Gβ (BraA.Gβ1, BraA.Gβ2, and BraA.Gβ3), and five Gγ (BraA.Gγ1, BraA.Gγ2, BraA.Gγ3, BraA.Gγ4, and BraA.Gγ5) genes from B. rapa, with a possibility of 15 Gαβγ heterotrimer combinations. Our analysis suggested that the process of genome triplication coupled with gene-loss (gene-fractionation) phenomenon have shaped the quantitative and sequence diversity of G-protein subunit genes in the extant B. rapa genome. Detailed expression analysis using qRT-PCR assays revealed that the G-protein genes have retained ubiquitous but distinct expression profiles across plant development. The expression of multiple G-protein genes was differentially regulated during seed-maturation and germination stages, and in response to various phytohormone treatments and stress conditions. Yeast-based interaction analysis showed that G-protein subunits interacted in most of the possible combinations, with some degree of subunit-specific interaction specificity, to control the functional selectivity of G-protein heterotrimer in different cell and tissue-types or in response to different environmental conditions. Taken together, this research identifies a highly diverse G-protein signaling network known to date from B. rapa, and provides a clue about the possible complexity of G-protein signaling networks present across globally important Brassica species.

  17. Evolution, Expression Differentiation and Interaction Specificity of Heterotrimeric G-Protein Subunit Gene Family in the Mesohexaploid Brassica rapa

    PubMed Central

    Arya, Gulab C.; Kumar, Roshan; Bisht, Naveen C.

    2014-01-01

    Heterotrimeric G-proteins, comprising of Gα, Gβ, and Gγ subunits, are important signal transducers which regulate many aspects of fundamental growth and developmental processes in all eukaryotes. Initial studies in model plants Arabidopsis and rice suggest that the repertoire of plant G-protein is much simpler than that observed in metazoans. In order to assess the consequence of whole genome triplication events within Brassicaceae family, we investigated the multiplicity of G-protein subunit genes in mesohexaploid Brassica rapa, a globally important vegetable and oilseed crop. We identified one Gα (BraA.Gα1), three Gβ (BraA.Gβ1, BraA.Gβ2, and BraA.Gβ3), and five Gγ (BraA.Gγ1, BraA.Gγ2, BraA.Gγ3, BraA.Gγ4, and BraA.Gγ5) genes from B. rapa, with a possibility of 15 Gαβγ heterotrimer combinations. Our analysis suggested that the process of genome triplication coupled with gene-loss (gene-fractionation) phenomenon have shaped the quantitative and sequence diversity of G-protein subunit genes in the extant B. rapa genome. Detailed expression analysis using qRT-PCR assays revealed that the G-protein genes have retained ubiquitous but distinct expression profiles across plant development. The expression of multiple G-protein genes was differentially regulated during seed-maturation and germination stages, and in response to various phytohormone treatments and stress conditions. Yeast-based interaction analysis showed that G-protein subunits interacted in most of the possible combinations, with some degree of subunit-specific interaction specificity, to control the functional selectivity of G-protein heterotrimer in different cell and tissue-types or in response to different environmental conditions. Taken together, this research identifies a highly diverse G-protein signaling network known to date from B. rapa, and provides a clue about the possible complexity of G-protein signaling networks present across globally important Brassica species. PMID

  18. Characterization of protein-protein interaction interfaces from a single species.

    PubMed

    Talavera, David; Robertson, David L; Lovell, Simon C

    2011-01-01

    Most proteins attain their biological functions through specific interactions with other proteins. Thus, the study of protein-protein interactions and the interfaces that mediate these interactions is of prime importance for the understanding of biological function. In particular the precise determinants of binding specificity and their contributions to binding energy within protein interfaces are not well understood. In order to better understand these determinants an appropriate description of the interaction surface is needed. Available data from the yeast Saccharomyces cerevisiae allow us to focus on a single species and to use all the available structures, correcting for redundancy, instead of using structural representatives. This allows us to control for potentially confounding factors that may affect sequence propensities. We find a significant contribution of main-chain atoms to protein-protein interactions. These include interactions both with other main-chain and side-chain atoms on the interacting chain. We find that the type of interaction depends on both amino acid and secondary structure type involved in the contact. For example, residues in α-helices and large amino acids are the most likely to be involved in interactions through their side-chain atoms. We find an intriguing homogeneity when calculating the average solvation energy of different areas of the protein surface. Unexpectedly, homo- and hetero-complexes have quite similar results for all analyses. Our findings demonstrate that the manner in which protein-protein interactions are formed is determined by the residue type and the secondary structure found in the interface. However the homogeneity of the desolvation energy despite heterogeneity of interface properties suggests a complex relationship between interface composition and binding energy.

  19. Hydrogen ion titration of 12 S rape seed protein and partial N-terminal sequence of one of it's subunits.

    PubMed

    Bhushan, R; Mahesh, V K; Mallikharjun, P V

    1989-10-01

    The high molecular weight 12 S protein from rape seed was isolated in a homogeneous form and characterized. Six subunits were isolated by PAGE in the presence of SDS and 0.2 M 2-mercaptoethanol. These subunits (s1 to s6) were found in the protein in the weight ratio of 1.32:1.2:1.15:1.0:1.21:1.11. The molecular weights and first two N-terminal amino acids of the isolated subunits were 64,800 and phenylalanine, alanine (s1), 50,650 and valine, tyrosine (s2), 42,500 and phenylalanine, leucine (s3), 28,800 and threonine, glutamic acid (s4), 19,100 and cystine, isoleucine (s5) and 15,600 and alanine, phenylalanine (s6). The number of side chain carboxyl, imidazole and epsilon-amino groups were calculated from the hydrogen ion titrations, which were in agreement with the amino acid assay. Besides, the N-terminal amino acid sequence upto 43 residues for one subunit (s6) is reported using Edman degradation.

  20. An atypical heterotrimeric G-protein γ-subunit is involved in guard cell K⁺-channel regulation and morphological development in Arabidopsis thaliana.

    PubMed

    Chakravorty, David; Trusov, Yuri; Zhang, Wei; Acharya, Biswa R; Sheahan, Michael B; McCurdy, David W; Assmann, Sarah M; Botella, José Ramón

    2011-09-01

    Currently, there are strong inconsistencies in our knowledge of plant heterotrimeric G-proteins that suggest the existence of additional members of the family. We have identified a new Arabidopsis G-protein γ-subunit (AGG3) that modulates morphological development and ABA-regulation of stomatal aperture. AGG3 strongly interacts with the Arabidopsis G-protein β-subunit in vivo and in vitro. Most importantly, AGG3-deficient mutants account for all but one of the 'orphan' phenotypes previously unexplained by the two known γ-subunits in Arabidopsis. AGG3 has unique characteristics never before observed in plant or animal systems, such as its size (more than twice that of canonical γ-subunits) and the presence of a C-terminal Cys-rich domain. AGG3 thus represent a novel class of G-protein γ-subunits, widely spread throughout the plant kingdom but not present in animals. Homologues of AGG3 in rice have been identified as important quantitative trait loci for grain size and yield, but due to the atypical nature of the proteins their identity as G-protein subunits was thus far unknown. Our work demonstrates a similar trend in seeds of Arabidopsis agg3 mutants, and implicates G-proteins in such a crucial agronomic trait. The discovery of this highly atypical subunit reinforces the emerging notion that plant and animal G-proteins have distinct as well as shared evolutionary pathways.

  1. The Gα4 G protein subunit interacts with the MAP kinase ERK2 using a D-motif that regulates developmental morphogenesis in Dictyostelium

    PubMed Central

    Nguyen, Hoai-Nghia; Hadwiger, Jeffrey A.

    2009-01-01

    G proteinsubunits contribute to the specificity of different signal transduction pathways in Dictyostelium discoideum but Gα subunit-effector interactions have not been previously identified. The requirement of the Dictyostelium Gα4 subunit for MAP kinase (MAPK) activation and the identification of a putative MAPK docking site (D-motif) in this subunit suggested a possible interaction between the Gα4 subunit and MAPKs. In vivo association of the Gα4 subunit and ERK2 was demonstrated by pull-down and co-immunoprecipitation assays. Alteration of the D-motif reduced Gα4 subunit-ERK2 interactions but only slightly altered MAPK activation in response to folate. Expression of the Gα4 subunit with the altered D-motif in gα4− cells allowed for slug formation but not the morphogenesis associated with culmination. Expression of this mutant Gα4 subunit was sufficient to rescue chemotactic movement to folate. Alteration of the D-motif also reduced the aggregation defect associated with constitutively active Gα4 subunits. These results suggest Gα4 subunit-MAPK interactions are necessary for developmental morphogenesis but not for chemotaxis to folate. PMID:19765570

  2. Immunological identification of a cAMP-dependent protein kinase regulatory subunit-like protein from the Trypanosoma equiperdum TeAp-N/D1 isolate.

    PubMed

    Calabokis, Maritza; González, Yelvis; Merchán, Adriana; Escalona, José L; Araujo, Nelson A; Sanz-Rodríguez, Carlos E; Cywiak, Carolina; Spencer, Lilian M; Martínez, Juan C; Bubis, José

    2016-01-01

    Polyclonal immunoglobulin Y (IgY) antibodies were produced in chicken eggs against the purified R(II)-subunit of the cAMP-dependent protein kinase (PKA) from pig heart, which corresponds to the Sus scrofa R(II)α isoform. In order to evaluate whether Trypanosoma equiperdum possessed PKA R-like proteins, parasites from the Venezuelan TeAp-N/D1 strain were examined using the generated anti-R(II) IgY antibodies. Western blot experiments revealed a 57-kDa polypeptide band that was distinctively recognized by these antibodies. Likewise, polyclonal antibodies raised in mice ascites against the recombinant T. equiperdum PKA R-like protein recognized the PKA R(II)-subunit purified from porcine heart and the recombinant human PKA R(I)β-subunit by immunoblotting. However, a partially purified fraction of the parasite PKA R-like protein was not capable of binding cAMP, implying that this protein is not a direct downstream cAMP effector in T. equiperdum. Although the function of the S. scrofa PKA R(II)α and the T. equiperdum PKA R-like protein appear to be different, their cross-reactivity together with results obtained by bioinformatics techniques corroborated the high level of homology exhibited by both proteins. Moreover, its presence in other trypanosomatids suggests an important cellular role of PKA R-like proteins in parasite physiology.

  3. Functional protein expression of multiple sodium channel alpha- and beta-subunit isoforms in neonatal cardiomyocytes.

    PubMed

    Kaufmann, Susann G; Westenbroek, Ruth E; Zechner, Christoph; Maass, Alexander H; Bischoff, Sebastian; Muck, Jenny; Wischmeyer, Erhard; Scheuer, Todd; Maier, Sebastian K G

    2010-01-01

    Voltage-gated sodium channels are composed of pore-forming alpha- and auxiliary beta-subunits and are responsible for the rapid depolarization of cardiac action potentials. Recent evidence indicates that neuronal tetrodotoxin (TTX) sensitive sodium channel alpha-subunits are expressed in the heart in addition to the predominant cardiac TTX-resistant Na(v)1.5 sodium channel alpha-subunit. These TTX-sensitive isoforms are preferentially localized in the transverse tubules of rodents. Since neonatal cardiomyocytes have yet to develop transverse tubules, we determined the complement of sodium channel subunits expressed in these cells. Neonatal rat ventricular cardiomyocytes were stained with antibodies specific for individual isoforms of sodium channel alpha- and beta-subunits. alpha-actinin, a component of the z-line, was used as an intracellular marker of sarcomere boundaries. TTX-sensitive sodium channel alpha-subunit isoforms Na(v)1.1, Na(v)1.2, Na(v)1.3, Na(v)1.4 and Na(v)1.6 were detected in neonatal rat heart but at levels reduced compared to the predominant cardiac alpha-subunit isoform, Na(v)1.5. Each of the beta-subunit isoforms (beta1-beta4) was also expressed in neonatal cardiac cells. In contrast to adult cardiomyocytes, the alpha-subunits are distributed in punctate clusters across the membrane surface of neonatal cardiomyocytes; no isoform-specific subcellular localization is observed. Voltage clamp recordings in the absence and presence of 20 nM TTX provided functional evidence for the presence of TTX-sensitive sodium current in neonatal ventricular myocardium which represents between 20 and 30% of the current, depending on membrane potential and experimental conditions. Thus, as in the adult heart, a range of sodium channel alpha-subunits are expressed in neonatal myocytes in addition to the predominant TTX-resistant Na(v)1.5 alpha-subunit and they contribute to the total sodium current.

  4. Optical control of endogenous proteins with a photoswitchable conditional subunit reveals a role for TREK1 in GABA(B) signaling.

    PubMed

    Sandoz, Guillaume; Levitz, Joshua; Kramer, Richard H; Isacoff, Ehud Y

    2012-06-21

    Selective ligands are lacking for many neuronal signaling proteins. Photoswitched tethered ligands (PTLs) have enabled fast and reversible control of specific proteins containing a PTL anchoring site and have been used to remote control overexpressed proteins. We report here a scheme for optical remote control of native proteins using a "photoswitchable conditional subunit" (PCS), which contains the PTL anchoring site as well as a mutation that prevents it from reaching the plasma membrane. In cells lacking native subunits for the protein, the PCS remains nonfunctional internally. However, in cells expressing native subunits, the native subunit and PCS coassemble, traffic to the plasma membrane, and place the native protein under optical control provided by the coassembled PCS. We apply this approach to the TREK1 potassium channel, which lacks selective, reversible blockers. We find that TREK1, typically considered to be a leak channel, contributes to the hippocampal GABA(B) response.

  5. Protein kinase C epsilon activates lens mitochondrial cytochrome c oxidase subunit IV during hypoxia.

    PubMed

    Barnett, Michael; Lin, Dingbo; Akoyev, Vladimir; Willard, Lloyd; Takemoto, Dolores

    2008-02-01

    Protein kinase C (PKC) isoforms have been identified as major cellular signaling proteins that act directly in response to oxidation conditions. In retina and lens two isoforms of PKC respond to changes in oxidative stress, PKCgamma and PKCepsilon, while only PKCepsilon is found in heart. In heart the PKCepsilon acts on connexin 43 to protect from hypoxia. The presence of both isoforms in the lens led to this study to determine if lens PKCepsilon had unique targets. Both lens epithelial cells in culture and whole mouse lens were examined using PKC isoform-specific enzyme activity assays, co-immunoprecipitation, confocal microscopy, immunoblots, and light and electron microscopy. PKCepsilon was found in lens epithelium and cortex but not in the nucleus of mouse lens. The PKCepsilon isoform was activated in both epithelium and whole lens by 5% oxygen when compared to activity at 21% oxygen. In hypoxic conditions (5% oxygen) the PKCepsilon co-immunoprecipitated with the mitochondrial cytochrome c oxidase IV subunit (CytCOx). Concomitant with this the CytCOx enzyme activity was elevated and increased co-localization of CytCOx with PCKvarepsilon was observed using immunolabeling and confocal microscopy. In contrast, no hypoxia-induced activation of CytCOx was observed in lenses from the PKCepsilon knockout mice. Lens from 6-week-old PKCepsilon knockout mice had a disorganized bow region which was filled with vacuoles indicating a possible loss of mitochondria but the size of the lens was not altered. Electron microscopy demonstrated that the nuclei of the PCKepsilon knockout mice were abnormal in shape. Thus, PKCepsilon is found to be activated by hypoxia and this results in the activation of the mitochondrial protein CytCOx. This could protect the lens from mitochondrial damage under the naturally hypoxic conditions observed in this tissue. Lens oxygen levels must remain low. Elevation of oxygen which occurs during vitreal detachment or liquification is associated with

  6. Different expression of protein kinase A (PKA) regulatory subunits in cortisol-secreting adrenocortical tumors: Relationship with cell proliferation

    SciTech Connect

    Mantovani, G.; Lania, A.G.; Bondioni, S.; Peverelli, E.; Pedroni, C.; Ferrero, S.; Pellegrini, C.; Vicentini, L.; Arnaldi, G.; Bosari, S.; Beck-Peccoz, P.; Spada, A.

    2008-01-01

    The four regulatory subunits (R1A, R1B, R2A, R2B) of protein kinase A (PKA) are differentially expressed in several cancer cell lines and exert distinct roles in growth control. Mutations of the R1A gene have been found in patients with Carney complex and in a minority of sporadic primary pigmented nodular adrenocortical disease (PPNAD). The aim of this study was to evaluate the expression of PKA regulatory subunits in non-PPNAD adrenocortical tumors causing ACTH-independent Cushing's syndrome and to test the impact of differential expression of these subunits on cell growth. Immunohistochemistry demonstrated a defective expression of R2B in all cortisol-secreting adenomas (n = 16) compared with the normal counterpart, while both R1A and R2A were expressed at high levels in the same tissues. Conversely, carcinomas (n = 5) showed high levels of all subunits. Sequencing of R1A and R2B genes revealed a wild type sequence in all tissues. The effect of R1/R2 ratio on proliferation was assessed in mouse adrenocortical Y-1 cells. The R2-selective cAMP analogue 8-Cl-cAMP dose-dependently inhibited Y-1 cell proliferation and induced apoptosis, while the R1-selective cAMP analogue 8-HA-cAMP stimulated cell proliferation. Finally, R2B gene silencing induced up-regulation of R1A protein, associated with an increase in cell proliferation. In conclusion, we propose that a high R1/R2 ratio favors the proliferation of well differentiated and hormone producing adrenocortical cells, while unbalanced expression of these subunits is not required for malignant transformation.

  7. Identification of single nucleotide polymorphism in protein phosphatase 1 regulatory subunit 11 gene in Murrah bulls

    PubMed Central

    Jain, Varsha; Patel, Brijesh; Umar, Farhat Paul; Ajithakumar, H. M.; Gurjar, Suraj K.; Gupta, I. D.; Verma, Archana

    2017-01-01

    Aim: This study was conducted with the objective to identify single nucleotide polymorphism (SNP) in protein phosphatase 1 regulatory subunit 11 (PPP1R11) gene in Murrah bulls. Materials and Methods: Genomic DNA was isolated by phenol–chloroform extraction method from the frozen semen samples of 65 Murrah bulls maintained at Artificial Breeding Research Centre, ICAR-National Dairy Research Institute, Karnal. The quality and concentration of DNA was checked by spectrophotometer reading and agarose gel electrophoresis. The target region of PPP1R11 gene was amplified using four sets of primer designed based on Bos taurus reference sequence. The amplified products were sequenced and aligned using Clustal Omega for identification of SNPs. Animals were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using EcoNI restriction enzyme. Results: The sequences in the NCBI accession number NW_005785016.1 for Bubalus bubalis were compared and aligned with the edited sequences of Murrah bulls with Clustal Omega software. A total of 10 SNPs were found, out of which 1 at 5’UTR, 3 at intron 1, and 6 at intron 2 region. PCR-RFLP using restriction enzyme EcoNI revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study. Conclusion: A total of 10 SNPs were found. PCR-RFLP revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study, due to which association analysis with conception rate was not feasible. PMID:28344410

  8. A strategy based on protein-protein interface motifs may help in identifying drug off-targets.

    PubMed

    Engin, H Billur; Keskin, Ozlem; Nussinov, Ruth; Gursoy, Attila

    2012-08-27

    Networks are increasingly used to study the impact of drugs at the systems level. From the algorithmic standpoint, a drug can "attack" nodes or edges of a protein-protein interaction network. In this work, we propose a new network strategy, "The Interface Attack", based on protein-protein interfaces. Similar interface architectures can occur between unrelated proteins. Consequently, in principle, a drug that binds to one has a certain probability of binding to others. The interface attack strategy simultaneously removes from the network all interactions that consist of similar interface motifs. This strategy is inspired by network pharmacology and allows inferring potential off-targets. We introduce a network model that we call "Protein Interface and Interaction Network (P2IN)", which is the integration of protein-protein interface structures and protein interaction networks. This interface-based network organization clarifies which protein pairs have structurally similar interfaces and which proteins may compete to bind the same surface region. We built the P2IN with the p53 signaling network and performed network robustness analysis. We show that (1) "hitting" frequent interfaces (a set of edges distributed around the network) might be as destructive as eleminating high degree proteins (hub nodes), (2) frequent interfaces are not always topologically critical elements in the network, and (3) interface attack may reveal functional changes in the system better than the attack of single proteins. In the off-target detection case study, we found that drugs blocking the interface between CDK6 and CDKN2D may also affect the interaction between CDK4 and CDKN2D.

  9. A chemical biology approach demonstrates G protein βγ subunits are sufficient to mediate directional neutrophil chemotaxis.

    PubMed

    Surve, Chinmay R; Lehmann, David; Smrcka, Alan V

    2014-06-20

    Our laboratory has identified a number of small molecules that bind to G protein βγ subunits (Gβγ) by competing for peptide binding to the Gβγ "hot spot." M119/Gallein were identified as inhibitors of Gβγ subunit signaling. Here we examine the activity of another molecule identified in this screen, 12155, which we show that in contrast to M119/Gallein had no effect on Gβγ-mediated phospholipase C or phosphoinositide 3-kinase (PI3K) γ activation in vitro. Also in direct contrast to M119/Gallein, 12155 caused receptor-independent Ca(2+) release, and activated other downstream targets of Gβγ including extracellular signal regulated kinase (ERK), protein kinase B (Akt) in HL60 cells differentiated to neutrophils. We show that 12155 releases Gβγ in vitro from Gαi1β1γ2 heterotrimers by causing its dissociation from GαGDP without inducing nucleotide exchange in the Gα subunit. We used this novel probe to examine the hypothesis that Gβγ release is sufficient to direct chemotaxis of neutrophils in the absence of receptor or G protein α subunit activation. 12155 directed chemotaxis of HL60 cells and primary neutrophils in a transwell migration assay with responses similar to those seen for the natural chemotactic peptide n-formyl-Met-Leu-Phe. These data indicate that release of free Gβγ is sufficient to drive directional chemotaxis in a G protein-coupled receptor signaling-independent manner.

  10. Mutations in exocyst complex subunit SEC6 gene impaired polar auxin transport and PIN protein recycling in Arabidopsis primary root.

    PubMed

    Tan, Xiaoyun; Feng, Yihong; Liu, Yulong; Bao, Yiqun

    2016-09-01

    Polar auxin transport, which is critical for land plant pattern formation and directional growth, is largely depended on asymmetric distribution of PIN proteins at the plasma membrane (PM). Endocytosis and recycling processes play important roles in regulating PIN protein distribution and abundance at the PM. Two subunits (SEC8, EXO70A1) of exocyst, an octameric vesicle-tethering complex, have been reported to be involved in PIN protein recycling in Arabidopsis. However, the function of exocyst complex in PIN protein recycling and polar auxin transport remains incompletely understood. In this study, we utilized two SEC6 down-regulation mutants (PRsec6-1 and PRsec6-2) to investigate the role of exocyst subunit SEC6 in the primary root development, polar auxin transport and PIN proteins recycling. We found that in PRsec6 mutants: 1. Primary root growth was retarded, and lateral root initiation were compromised. 2. Primary roots were sensitive to exogenous auxin 1-napthalene acetic acid (NAA) but not 2,4-dichlorophenoxy (2.4-D). 3. Recycling of PIN1 and PIN2 proteins from the Brefeldin A (BFA) compartment to the PM was delayed. 4. Vesicles accumulated in the primary root tip cells, especially accumulated in the cytosol closed to the PM. These results further demonstrated that the exocyst complex plays an important role in PIN protein recycling and polar auxin transport in Arabidopsis primary root.

  11. Uncovering the structural basis of protein interactions with efficient clustering of 3-D interaction interfaces.

    PubMed

    Aung, Z; Tan, S-H; Ng, S-K; Tan, K-L

    2007-01-01

    The biological mechanisms with which proteins interact with one another are best revealed by studying the structural interfaces between interacting proteins. Protein-protein interfaces can be extracted from 3-D structural data of protein complexes and then clustered to derive biological insights. However, conventional protein interface clustering methods lack computational scalability and statistical support. In this work, we present a new method named "PPiClust" to systematically encode, cluster and analyze similar 3-D interface patterns in protein complexes efficiently. Experimental results showed that our method is effective in discovering visually consistent and statistically significant clusters of interfaces, and at the same time sufficiently time-efficient to be performed on a single computer. The interface clusters are also useful for uncovering the structural basis of protein interactions. Analysis of the resulting interface clusters revealed groups of structurally diverse proteins having similar interface patterns. We also found, in some of the interface clusters, the presence of well-known linear binding motifs which were non-contiguous in the primary sequences. These results suggest that PPiClust can discover not only statistically significant but also biologically significant protein interface clusters from protein complex structural data.

  12. Overproduction of the regulatory subunit of the cAMP-dependent protein kinase blocks the differentiation of Dictyostelium discoideum.

    PubMed Central

    Simon, M N; Driscoll, D; Mutzel, R; Part, D; Williams, J; Véron, M

    1989-01-01

    During the aggregation of Dictyostelium discoideum extracellular cAMP is known to act as a chemotractant and as an inducer of cellular differentiation. However, its intracellular role as a second messenger remains obscure. We have constructed a fusion gene consisting of the cDNA encoding the regulatory subunit (R) of the cAMP-dependent protein kinase fused to the promoter and N-terminal-proximal sequences of a Dictyostelium actin gene. Stable transformants, containing multiple copies of this gene, overproduce the R subunit which accumulates prematurely relative to the endogenous protein. These transformants fail to aggregate. Detailed analysis has shown that they are blocked at interphase, the period prior to aggregation, and that they are severely defective in most responses to cAMP including the induction of gene expression. Our observations suggest that intracellular cAMP acts, presumably by activation of the catalytic subunit of the cAMP-dependent protein kinase, to facilitate early development. Images PMID:2551673

  13. Roles of Replication Protein-a Subunits 2 and 3 in DNA Replication Fork Movement in Saccharomyces Cerevisiae

    PubMed Central

    Maniar, H. S.; Wilson, R.; Brill, S. J.

    1997-01-01

    Replication Protein-A, the eukaryotic SSB, consists of a large subunit (RPA1) with strong ssDNA binding activity and two smaller subunits (RPA2 and 3) that may cooperate with RPA1 to bind ssDNA in a higher-order mode. To determine the in vivo function of the two smaller subunits and the potential role of higher-order ssDNA binding, we isolated an assortment of heat-lethal mutations in the genes encoding RPA2 and RPA3. At the permissive temperature, the mutants show a range of effects on DNA replication fidelity and sensitivities to UV and MMS. At the nonpermissive temperature, four out of five RPA2 mutants show a fast-stop DNA synthesis phenotype typical of a replication fork block. In contrast, the fifth RPA2 mutant and all RPA3 mutants are able to complete at least one round of DNA replication at the nonpermissive temperature. The effect of these mutations on the stability of the RPA complex was tested using a coprecipitation assay. At the nonpermissive temperature, we find that RPA1 and RPA2 are dissociated in the fast-stop mutants, but not in the slow-stop mutants. Thus, replication fork movement in vivo requires the association of at least two subunits of RPA. This result is consistent with the hypothesis that RPA functions in vivo by binding ssDNA in a higher-order mode. PMID:9093844

  14. Downregulation of GABA[Subscript A] Receptor Protein Subunits a6, ß2, d, e, ?2, ?, and ?2 in Superior Frontal Cortex of Subjects with Autism

    ERIC Educational Resources Information Center

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rustan, Oyvind G.; Rooney, Robert J.; Thuras, Paul D.

    2014-01-01

    We measured protein and mRNA levels for nine gamma-aminobutyric acid A (GABA[subscript A]) receptor subunits in three brain regions (cerebellum, superior frontal cortex, and parietal cortex) in subjects with autism versus matched controls. We observed changes in mRNA for a number of GABA[subscript A] and GABA[subscript B] subunits and overall…

  15. The beta subunit of the Drosophila melanogaster ATP synthase: cDNA cloning, amino acid analysis and identification of the protein in adult flies.

    PubMed

    Peña, P; Garesse, R

    1993-09-15

    The cDNA encoding the Drosophila melanogaster beta subunit of H+ ATP synthase has been cloned and sequenced. The predicted mature protein is highly homologous to the equivalent beta subunits of other organisms and is preceded by a signal peptide of 31 amino acids, that although not conserved at primary sequence level has the characteristics of leader peptides present in other mitochondrial proteins. We have raised polyclonal antibodies that specifically recognize the beta H+ ATP synthase subunit present in Drosophila melanogaster protein extracts. This is the first time that a gene of the ATP synthase complex has been characterized in the invertebrate phyla.

  16. Template-based identification of protein-protein interfaces using eFindSitePPI.

    PubMed

    Maheshwari, Surabhi; Brylinski, Michal

    2016-01-15

    Protein-protein interactions orchestrate virtually all cellular processes, therefore, their exhaustive exploration is essential for the comprehensive understanding of cellular networks. A reliable identification of interfacial residues is vital not only to infer the function of individual proteins and their assembly into biological complexes, but also to elucidate the molecular and physicochemical basis of interactions between proteins. With the exponential growth of protein sequence data, computational approaches for detecting protein interface sites have drawn an increased interest. In this communication, we discuss the major features of eFindSite(PPI), a recently developed template-based method for interface residue prediction available at http://brylinski.cct.lsu.edu/efindsiteppi. We describe the requirements and installation procedures for the stand-alone version, and explain the content and format of output data. Furthermore, the functionality of the eFindSite(PPI) web application that is designed to provide a simple and convenient access for the scientific community is presented with illustrative examples. Finally, we discuss common problems encountered in predicting protein interfaces and set forth directions for the future development of eFindSite(PPI).

  17. Collision-induced release, ion mobility separation, and amino acid sequence analysis of subunits from mass-selected noncovalent protein complexes.

    PubMed

    Rathore, Deepali; Dodds, Eric D

    2014-09-01

    In recent years, mass spectrometry has become a valuable tool for detecting and characterizing protein-protein interactions and for measuring the masses and subunit stoichiometries of noncovalent protein complexes. The gas-phase dissociation of noncovalent protein assemblies via tandem mass spectrometry can be useful in confirming subunit masses and stoichiometries; however, dissociation experiments that are able to yield subunit sequence information must usually be conducted separately. Here, we furnish proof of concept for a method that allows subunit sequence information to be directly obtained from a protein aggregate in a single gas-phase analysis. The experiments were carried out using a quadrupole time-of-flight mass spectrometer equipped with a traveling-wave ion mobility separator. This instrument configuration allows for a noncovalent protein assembly to be quadrupole selected, then subjected to two successive rounds of collision-induced dissociation with an intervening stage of ion mobility separation. This approach was applied to four model proteins as their corresponding homodimers: glucagon, ubiquitin, cytochrome c, and β-lactoglobulin. In each case, b- and y-type fragment ions were obtained upon further collisional activation of the collisionally-released subunits, resulting in up to 50% sequence coverage. Owing to the incorporation of an ion mobility separation, these results also suggest the intriguing possibility of measuring complex mass, complex collisional cross section, subunit masses, subunit collisional cross sections, and sequence information for the subunits in a single gas-phase experiment. Overall, these findings represent a significant contribution towards the realization of protein interactomic analyses, which begin with native complexes and directly yield subunit identities.

  18. Collision-Induced Release, Ion Mobility Separation, and Amino Acid Sequence Analysis of Subunits from Mass-Selected Noncovalent Protein Complexes

    NASA Astrophysics Data System (ADS)

    Rathore, Deepali; Dodds, Eric D.

    2014-09-01

    In recent years, mass spectrometry has become a valuable tool for detecting and characterizing protein-protein interactions and for measuring the masses and subunit stoichiometries of noncovalent protein complexes. The gas-phase dissociation of noncovalent protein assemblies via tandem mass spectrometry can be useful in confirming subunit masses and stoichiometries; however, dissociation experiments that are able to yield subunit sequence information must usually be conducted separately. Here, we furnish proof of concept for a method that allows subunit sequence information to be directly obtained from a protein aggregate in a single gas-phase analysis. The experiments were carried out using a quadrupole time-of-flight mass spectrometer equipped with a traveling-wave ion mobility separator. This instrument configuration allows for a noncovalent protein assembly to be quadrupole selected, then subjected to two successive rounds of collision-induced dissociation with an intervening stage of ion mobility separation. This approach was applied to four model proteins as their corresponding homodimers: glucagon, ubiquitin, cytochrome c, and β-lactoglobulin. In each case, b- and y-type fragment ions were obtained upon further collisional activation of the collisionally-released subunits, resulting in up to 50% sequence coverage. Owing to the incorporation of an ion mobility separation, these results also suggest the intriguing possibility of measuring complex mass, complex collisional cross section, subunit masses, subunit collisional cross sections, and sequence information for the subunits in a single gas-phase experiment. Overall, these findings represent a significant contribution towards the realization of protein interactomic analyses, which begin with native complexes and directly yield subunit identities.

  19. Unique domain anchoring of Src to synaptic NMDA receptors via the mitochondrial protein NADH dehydrogenase subunit 2

    PubMed Central

    Gingrich, Jeffrey R.; Pelkey, Kenneth A.; Fam, Sami R.; Huang, Yueqiao; Petralia, Ronald S.; Wenthold, Robert J.; Salter, Michael W.

    2004-01-01

    Src is the prototypic protein tyrosine kinase and is critical for controlling diverse cellular functions. Regions in Src define structural and functional domains conserved in many cell signaling proteins. Src also contains a region of low sequence conservation termed the unique domain, the function of which has until now remained enigmatic. Here, we show that the unique domain of Src is a protein–protein interaction region and we identify NADH dehydrogenase subunit 2 (ND2) as a Src unique domain-interacting protein. ND2 is a subunit of complex I in mitochondria, but we find that ND2 interacts with Src outside this organelle at excitatory synapses in the brain. ND2 acts as an adapter protein anchoring Src to the N-methyl-d-aspartate (NMDA) receptor complex, and is crucial for Src regulation of synaptic NMDA receptor activity. By showing an extramitochondrial action for a protein encoded in the mitochondrial genome, we identify a previously unsuspected means by which mitochondria regulate cellular function, suggesting a new paradigm that may be of general relevance for control of Src signaling. PMID:15069201

  20. Transport of the GlcNAc-1-phosphotransferase α/β-subunit precursor protein to the Golgi apparatus requires a combinatorial sorting motif.

    PubMed

    Franke, Mine; Braulke, Thomas; Storch, Stephan

    2013-01-11

    The Golgi-resident N-acetylglucosamine-1-phosphotransferase (PT) complex is composed of two α-, β-, and γ-subunits and represents the key enzyme for the biosynthesis of mannose 6-phosphate recognition marker on soluble lysosomal proteins. Mutations in the PT complex cause the lysosomal storage diseases mucolipidosis II and III. A prerequisite for the enzymatic activity is the site-1 protease-mediated cleavage of the PT α/β-subunit precursor protein in the Golgi apparatus. Here, we have investigated structural requirements of the PT α/β-subunit precursor protein for its efficient export from the endoplasmic reticulum (ER). Both wild-type and a cleavage-resistant type III membrane PT α/β-subunit precursor protein are exported whereas coexpressed separate α- and β-subunits failed to reach the cis-Golgi compartment. Mutational analyses revealed combinatorial, non-exchangeable dileucine and dibasic motifs located in a defined sequence context in the cytosolic N- and C-terminal domains that are required for efficient ER exit and subsequent proteolytic activation of the α/β-subunit precursor protein in the Golgi. In the presence of a dominant negative Sar1 mutant the ER exit of the PT α/β-subunit precursor protein is inhibited indicating its transport in coat protein complex II-coated vesicles. Expression studies of missense mutations identified in mucolipidosis III patients that alter amino acids in the N- and C-terminal domains demonstrated that the substitution of a lysine residue in close proximity to the dileucine sorting motif impaired ER-Golgi transport and subsequent activation of the PT α/β-subunit precursor protein. The data suggest that the oligomeric type III membrane protein PT complex requires a combinatorial sorting motif that forms a tertiary epitope to be recognized by distinct sites within the coat protein complex II machinery.

  1. A Machine Learning Approach for Hot-Spot Detection at Protein-Protein Interfaces

    PubMed Central

    Melo, Rita; Fieldhouse, Robert; Melo, André; Correia, João D. G.; Cordeiro, Maria Natália D. S.; Gümüş, Zeynep H.; Costa, Joaquim; Bonvin, Alexandre M. J. J.; Moreira, Irina S.

    2016-01-01

    Understanding protein-protein interactions is a key challenge in biochemistry. In this work, we describe a more accurate methodology to predict Hot-Spots (HS) in protein-protein interfaces from their native complex structure compared to previous published Machine Learning (ML) techniques. Our model is trained on a large number of complexes and on a significantly larger number of different structural- and evolutionary sequence-based features. In particular, we added interface size, type of interaction between residues at the interface of the complex, number of different types of residues at the interface and the Position-Specific Scoring Matrix (PSSM), for a total of 79 features. We used twenty-seven algorithms from a simple linear-based function to support-vector machine models with different cost functions. The best model was achieved by the use of the conditional inference random forest (c-forest) algorithm with a dataset pre-processed by the normalization of features and with up-sampling of the minor class. The method has an overall accuracy of 0.80, an F1-score of 0.73, a sensitivity of 0.76 and a specificity of 0.82 for the independent test set. PMID:27472327

  2. A Machine Learning Approach for Hot-Spot Detection at Protein-Protein Interfaces.

    PubMed

    Melo, Rita; Fieldhouse, Robert; Melo, André; Correia, João D G; Cordeiro, Maria Natália D S; Gümüş, Zeynep H; Costa, Joaquim; Bonvin, Alexandre M J J; Moreira, Irina S

    2016-07-27

    Understanding protein-protein interactions is a key challenge in biochemistry. In this work, we describe a more accurate methodology to predict Hot-Spots (HS) in protein-protein interfaces from their native complex structure compared to previous published Machine Learning (ML) techniques. Our model is trained on a large number of complexes and on a significantly larger number of different structural- and evolutionary sequence-based features. In particular, we added interface size, type of interaction between residues at the interface of the complex, number of different types of residues at the interface and the Position-Specific Scoring Matrix (PSSM), for a total of 79 features. We used twenty-seven algorithms from a simple linear-based function to support-vector machine models with different cost functions. The best model was achieved by the use of the conditional inference random forest (c-forest) algorithm with a dataset pre-processed by the normalization of features and with up-sampling of the minor class. The method has an overall accuracy of 0.80, an F1-score of 0.73, a sensitivity of 0.76 and a specificity of 0.82 for the independent test set.

  3. Quality control of a cytoplasmic protein complex: chaperone motors and the ubiquitin-proteasome system govern the fate of orphan fatty acid synthase subunit Fas2 of yeast.

    PubMed

    Scazzari, Mario; Amm, Ingo; Wolf, Dieter H

    2015-02-20

    For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2.

  4. A Determinant of Odorant Specificity Is Located at the Extracellular Loop 2-Transmembrane Domain 4 Interface of an Anopheles gambiae Odorant Receptor Subunit

    PubMed Central

    Hughes, David T.; Wang, Guirong; Zwiebel, Laurence J.

    2014-01-01

    To explore the structural basis for odorant specificity in odorant receptors of the human malaria vector mosquito, Anopheles gambiae, odorant-binding subunits (Agam\\Ors) expressed in Xenopus oocytes in combination with Agam\\Orco (coreceptor subunit) were assayed by 2-electrode voltage clamp against 25 structurally related odorants. Agam\\Or13 and Agam\\Or15 display 82% amino acid identity and had similar, but somewhat distinct odorant response profiles. The ratio of acetophenone to 4-methylphenol responses was used in a mutation-based analysis of Agam\\Or15, interchanging 37 disparate residues between Agam\\Or15 and Agam\\Or13. Eleven mutations caused significant changes in odorant responsiveness. Mutation of alanine 195 resulted in the largest shift in response ratio from Agam\\Or15 toward Agam\\Or13. Concentration–response analysis for a series of mutations of residue 195 revealed a large effect on acetophenone sensitivity, with EC50 values varying by >1800-fold and correlating with residue side chain length. Similar results were obtained for propiophenone and benzaldehyde. But, for other odorants, such as 4-methylphenol, 4-methylbenzaldehyde, and 4-methylpropiophenone, the effect of mutation was much smaller (EC50 values varied by ≤16-fold). These results show that alanine 195, putatively located at the second extracellular loop/fourth transmembrane domain interface, plays a critical role in determining the odorant response specificity of Agam\\Or15. PMID:25270378

  5. A determinant of odorant specificity is located at the extracellular loop 2-transmembrane domain 4 interface of an Anopheles gambiae odorant receptor subunit.

    PubMed

    Hughes, David T; Wang, Guirong; Zwiebel, Laurence J; Luetje, Charles W

    2014-11-01

    To explore the structural basis for odorant specificity in odorant receptors of the human malaria vector mosquito, Anopheles gambiae, odorant-binding subunits (Agam\\Ors) expressed in Xenopus oocytes in combination with Agam\\Orco (coreceptor subunit) were assayed by 2-electrode voltage clamp against 25 structurally related odorants. Agam\\Or13 and Agam\\Or15 display 82% amino acid identity and had similar, but somewhat distinct odorant response profiles. The ratio of acetophenone to 4-methylphenol responses was used in a mutation-based analysis of Agam\\Or15, interchanging 37 disparate residues between Agam\\Or15 and Agam\\Or13. Eleven mutations caused significant changes in odorant responsiveness. Mutation of alanine 195 resulted in the largest shift in response ratio from Agam\\Or15 toward Agam\\Or13. Concentration-response analysis for a series of mutations of residue 195 revealed a large effect on acetophenone sensitivity, with EC50 values varying by >1800-fold and correlating with residue side chain length. Similar results were obtained for propiophenone and benzaldehyde. But, for other odorants, such as 4-methylphenol, 4-methylbenzaldehyde, and 4-methylpropiophenone, the effect of mutation was much smaller (EC50 values varied by ≤16-fold). These results show that alanine 195, putatively located at the second extracellular loop/fourth transmembrane domain interface, plays a critical role in determining the odorant response specificity of Agam\\Or15.

  6. Rabies virus binding to the nicotinic acetylcholine receptor alpha subunit demonstrated by virus overlay protein binding assay.

    PubMed

    Gastka, M; Horvath, J; Lentz, T L

    1996-10-01

    A virus overlay protein binding assay was used to study binding of 125I-labelled rabies virus to the acetylcholine receptor (AChR) from Torpedo californica electric organ membranes. After gel electrophoresis of electric organ membranes and transfer of proteins to nitrocellulose, 125I-labelled alpha-bungarotoxin, a curaremimetic neurotoxin, bound to a 40 kDa band and 125I-labelled rabies virus bound to 51 kDa and 40 kDa bands. Binding of rabies virus to the 40 kDa band was inhibited by unlabelled alpha-bungarotoxin. In blots of affinity-purified AChR, labelled virus bound to the 40 kDa alpha subunit and was competed by alpha-bungarotoxin. Based on binding of rabies virus to the alpha subunit and the ability of alpha-bungarotoxin to compete for binding, rabies virus appears to bind to the neurotoxin-binding site of the nicotinic AChR alpha subunit.

  7. Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains.

    PubMed

    Shen, Peter S; Park, Joseph; Qin, Yidan; Li, Xueming; Parsawar, Krishna; Larson, Matthew H; Cox, James; Cheng, Yifan; Lambowitz, Alan M; Weissman, Jonathan S; Brandman, Onn; Frost, Adam

    2015-01-02

    In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo-electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein--not an mRNA--determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions ("CAT tails").

  8. Kinetics of the phosphotransferase reaction of the catalytic subunit of the tick salivary gland cAMP-dependent protein kinase

    SciTech Connect

    Mane, S.D.; Essenberg, R.C.; Sauer, J.R.

    1986-05-01

    The catalytic subunit of the cAMP dependent protein kinase was purified 100-fold from tick salivary glands. The enzyme mechanism of the phosphotransferase reaction catalyzed by this subunit was investigated. Highly purified enzyme did not show ATP-ase activity in the absence of protein substrates. Initial velocities were measured using histone H-1 or a synthetic heptapeptide, Kemptide, as P/sub i/ acceptors and (..gamma..-/sup 32/P) ATP as a phosphodonor. Patterns were consistent with a sequential, but not a ping pong mechanism. At high concentration (>2Km), histone showed substrate inhibition which was noncompetitive versus ATP. Product inhibition by Mg.ADP was competitive versus ATP and noncompetitive with respect to H-1. Phosphohistone on the other hand was noncompetitive with respect to H-1, but gave parabolic competitive inhibition against ATP. Dead-end inhibition by AMP-PNP, an analogue of ATP, was competitive and noncompetitive against ATP and H-1, respectively. The inhibitory of cAMP dependent protein kinase was noncompetitive with ATP and competitive with histone. These studies strongly suggest that the tick salivary gland protein kinase has a sequential mechanism with primarily ordered addition of ATP followed by protein substrate and ordered release of phosphoprotein and ADP, but some random character.

  9. A Nitrogen-Fixing Subunit Essential for Accumulating 4Fe-4S-Containing Photosystem I Core Proteins1[OPEN

    PubMed Central

    Nath, Krishna; Wessendorf, Ryan L.

    2016-01-01

    Nitrogen-fixation-subunit-U (NFU)-type proteins have been shown to be involved in the biogenesis of iron-sulfur clusters. We investigated the molecular function of a chloroplastic NFU-type iron-sulfur scaffold protein, NFU3, in Arabidopsis (Arabidopsis thaliana) using genetics approaches. Loss-of-function mutations in the NFU3 gene caused yellow pigmentation in leaves, reductions in plant size, leaf size, and growth rate, delay in flowering and seeding, and decreases in seed production. Biochemical and physiological analyses indicated that these defects are due to the substantial reductions in the abundances of 4Fe-4S-containing photosystem I (PSI) core subunits PsaA (where Psa stands for PSI), PsaB, and PsaC and a nearly complete loss of PSI activity. In addition to the substantial decreases in the amounts of PSI core proteins, the content of 3Fe-4S-containing ferredoxin-dependent glutamine oxoglutarate aminotransferases declined significantly in the nfu3 mutants. Furthermore, the absorption spectrum of the recombinant NFU3 protein showed features characteristic of 4Fe-4S and 3Fe-4S clusters, and the in vitro reconstitution experiment indicated an iron-sulfur scaffold function of NFU3. These data demonstrate that NFU3 is involved in the assembly and transfer of 4Fe-4S and 3Fe-4S clusters and that NFU3 is required for the accumulation of 4Fe-4S- and 3Fe-4S-containing proteins, especially 4Fe-4S-containing PSI core subunits, in the Arabidopsis chloroplast. PMID:27784767

  10. Small-Molecule Fusion Inhibitors Bind the pH-Sensing Stable Signal Peptide-GP2 Subunit Interface of the Lassa Virus Envelope Glycoprotein

    PubMed Central

    Shankar, Sundaresh; Whitby, Landon R.; Casquilho-Gray, Hedi E.; York, Joanne; Boger, Dale L.

    2016-01-01

    ABSTRACT Arenavirus species are responsible for severe life-threatening hemorrhagic fevers in western Africa and South America. Without effective antiviral therapies or vaccines, these viruses pose serious public health and biodefense concerns. Chemically distinct small-molecule inhibitors of arenavirus entry have recently been identified and shown to act on the arenavirus envelope glycoprotein (GPC) to prevent membrane fusion. In the tripartite GPC complex, pH-dependent membrane fusion is triggered through a poorly understood interaction between the stable signal peptide (SSP) and the transmembrane fusion subunit GP2, and our genetic studies have suggested that these small-molecule inhibitors act at this interface to antagonize fusion activation. Here, we have designed and synthesized photoaffinity derivatives of the 4-acyl-1,6-dialkylpiperazin-2-one class of fusion inhibitors and demonstrate specific labeling of both the SSP and GP2 subunits in a native-like Lassa virus (LASV) GPC trimer expressed in insect cells. Photoaddition is competed by the parental inhibitor and other chemically distinct compounds active against LASV, but not those specific to New World arenaviruses. These studies provide direct physical evidence that these inhibitors bind at the SSP-GP2 interface. We also find that GPC containing the uncleaved GP1-GP2 precursor is not susceptible to photo-cross-linking, suggesting that proteolytic maturation is accompanied by conformational changes at this site. Detailed mapping of residues modified by the photoaffinity adducts may provide insight to guide the further development of these promising lead compounds as potential therapeutic agents to treat Lassa hemorrhagic fever. IMPORTANCE Hemorrhagic fever arenaviruses cause lethal infections in humans and, in the absence of licensed vaccines or specific antiviral therapies, are recognized to pose significant threats to public health and biodefense. Lead small-molecule inhibitors that target the

  11. Isolation of photosystem II-enriched membranes and the oxygen-evolving complex subunit proteins from higher plants.

    PubMed

    Yamamoto, Yasusi; Leng, Jing; Shen, Jian-Ren

    2011-01-01

    We describe methods to isolate highly active oxygen-evolving photosystem II (PSII) membranes and core complexes from higher plants, and to purify subunits of the oxygen-evolving complex (OEC). The membrane samples used as the material for various in vitro studies of PSII are prepared by solubilizing thylakoid membranes with the nonionic detergent Triton X-100, and the core complexes are prepared by further solubilization of the PSII membranes with n-dodecyl-β-D-maltoside (β-DDM). The OEC subunit proteins are dissociated from the PSII-enriched membranes by alkaline or salt treatment, and are then purified by ion-exchange chromatography using an automated high performance liquid chromatography system.

  12. Functional identification of the promoter for the gene encoding the alpha subunit of calcium/calmodulin-dependent protein kinase II.

    PubMed Central

    Olson, N J; Massé, T; Suzuki, T; Chen, J; Alam, D; Kelly, P T

    1995-01-01

    To examine the expression of the alpha subunit of calcium/calmodulin-dependent protein kinase II, various 5' flanking genomic sequences were inserted into a chloramphenicol acetyltransferase (CAT) reporter plasmid and CAT enzyme activities were analyzed in transfected NB2a neuroblastoma cells and mRNA transcription was analyzed by nuclease protection assays. A core promoter was identified which contained an essential TATA element located 162 nt 5' to the transcription start site. Sequences 3' to the transcription start site, as well as 5' to the TATA element, increased levels of CAT activity in transfected cells. The alpha-subunit gene promoter displayed higher CAT activities, relative to a simian virus 40 promoter, in transfected neuronal cell lines than in nonneuronal cell lines. Results also suggested that sequence surrounding the natural alpha-gene transcription initiation site may be important for targeting transcription initiation 162 nt downstream of its TATA element. Images Fig. 1 Fig. 3 PMID:7878035

  13. A new strategy for protein interface identification using manifold learning method.

    PubMed

    Wang, Bing; Huang, De-Shuang; Jiang, Changjun

    2014-06-01

    Protein interactions play vital roles in biological processes. The study for protein interface will allow people to elucidate the mechanism of protein interaction. However, a large portion of protein interface data is incorrectly collected in current studies. In this paper, a novel strategy of dataset reconstruction using manifold learning method has been proposed for dealing with the noises in the interaction interface data whose definition is based on the residue distances among the different chains within protein complexes. Three support vector machine-based predictors are constructed using different protein features to identify the functional sites involved in the formation of protein interface. The experimental results achieved in this work demonstrate that our strategy can remove noises, and therefore improve the ability for identification of protein interfaces with 77.8% accuracy.

  14. Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis.

    PubMed

    Dresios, John; Aschrafi, Armaz; Owens, Geoffrey C; Vanderklish, Peter W; Edelman, Gerald M; Mauro, Vincent P

    2005-02-08

    The expression of Rbm3, a glycine-rich RNA-binding protein, is enhanced under conditions of mild hypothermia, and Rbm3 has been postulated to facilitate protein synthesis at colder temperatures. To investigate this possibility, Rbm3 was overexpressed as a c-Myc fusion protein in mouse neuroblastoma N2a cells. Cells expressing this fusion protein showed a 3-fold increase in protein synthesis at both 37 degrees C and 32 degrees C compared with control cells. Although polysome profiles of cells expressing the fusion protein and control cells were similar, several differences were noted, suggesting that Rbm3 might enhance the association of 40S and 60S ribosomal subunits at 32 degrees C. Studies to assess a direct interaction of Rbm3 with ribosomes showed that a fraction of Rbm3 was associated with 60S ribosomal subunits in an RNA-independent manner. It appeared unlikely that this association could explain the global enhancement of protein synthesis, however, because cells expressing the Rbm3 fusion protein showed no substantial increase in the size of their monosome and polysome peaks, suggesting that similar numbers of mRNAs were being translated at approximately the same rates. In contrast, a complex that sedimented between the top of the gradient and 40S subunits was less abundant in cells expressing recombinant Rbm3. Further analysis showed that the RNA component of this fraction was microRNA. We discuss the possibility that Rbm3 expression alters global protein synthesis by affecting microRNA levels and suggest that both Rbm3 and microRNAs are part of a homeostatic mechanism that regulates global levels of protein synthesis under normal and cold-stress conditions.

  15. Computational Simulation of the Activation Cycle of Gα Subunit in the G Protein Cycle Using an Elastic Network Model

    PubMed Central

    Kim, Min Hyeok; Kim, Young Jin; Kim, Hee Ryung; Jeon, Tae-Joon; Choi, Jae Boong; Chung, Ka Young; Kim, Moon Ki

    2016-01-01

    Agonist-activated G protein-coupled receptors (GPCRs) interact with GDP-bound G protein heterotrimers (Gαβγ) promoting GDP/GTP exchange, which results in dissociation of Gα from the receptor and Gβγ. The GTPase activity of Gα hydrolyzes GTP to GDP, and the GDP-bound Gα interacts with Gβγ, forming a GDP-bound G protein heterotrimer. The G protein cycle is allosterically modulated by conformational changes of the Gα subunit. Although biochemical and biophysical methods have elucidated the structure and dynamics of Gα, the precise conformational mechanisms underlying the G protein cycle are not fully understood yet. Simulation methods could help to provide additional details to gain further insight into G protein signal transduction mechanisms. In this study, using the available X-ray crystal structures of Gα, we simulated the entire G protein cycle and described not only the steric features of the Gα structure, but also conformational changes at each step. Each reference structure in the G protein cycle was modeled as an elastic network model and subjected to normal mode analysis. Our simulation data suggests that activated receptors trigger conformational changes of the Gα subunit that are thermodynamically favorable for opening of the nucleotide-binding pocket and GDP release. Furthermore, the effects of GTP binding and hydrolysis on mobility changes of the C and N termini and switch regions are elucidated. In summary, our simulation results enabled us to provide detailed descriptions of the structural and dynamic features of the G protein cycle. PMID:27483005

  16. Predicting the tolerated sequences for proteins and protein interfaces using RosettaBackrub flexible backbone design.

    PubMed

    Smith, Colin A; Kortemme, Tanja

    2011-01-01

    Predicting the set of sequences that are tolerated by a protein or protein interface, while maintaining a desired function, is useful for characterizing protein interaction specificity and for computationally designing sequence libraries to engineer proteins with new functions. Here we provide a general method, a detailed set of protocols, and several benchmarks and analyses for estimating tolerated sequences using flexible backbone protein design implemented in the Rosetta molecular modeling software suite. The input to the method is at least one experimentally determined three-dimensional protein structure or high-quality model. The starting structure(s) are expanded or refined into a conformational ensemble using Monte Carlo simulations consisting of backrub backbone and side chain moves in Rosetta. The method then uses a combination of simulated annealing and genetic algorithm optimization methods to enrich for low-energy sequences for the individual members of the ensemble. To emphasize certain functional requirements (e.g. forming a binding interface), interactions between and within parts of the structure (e.g. domains) can be reweighted in the scoring function. Results from each backbone structure are merged together to create a single estimate for the tolerated sequence space. We provide an extensive description of the protocol and its parameters, all source code, example analysis scripts and three tests applying this method to finding sequences predicted to stabilize proteins or protein interfaces. The generality of this method makes many other applications possible, for example stabilizing interactions with small molecules, DNA, or RNA. Through the use of within-domain reweighting and/or multistate design, it may also be possible to use this method to find sequences that stabilize particular protein conformations or binding interactions over others.

  17. Expression of a foreign Rubisco small subunit in tobacco with reduced levels of the native protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cDNA, ArRbcS3, for the small subunit of Rubisco from Amaranthus retroflexus (pigweed) was expressed in tobacco (Nicotiana tabacum) under the control of a strong leaf-specific Lhcb promoter. The coding region of the ArRbcS3 was fused to the plastid targeting sequence of the native tobacco rbcS to...

  18. Phosphorylation of chloroplast ribulose bisphosphate carboxylase/oxygenase small subunit by an envelope-bound protein kinase in situ.

    PubMed

    Soll, J; Buchanan, B B

    1983-06-10

    A new protein kinase of the cAMP independent type was found to be bound to the outer envelope membrane of spinach chloroplasts. While stimulated by Mg2+ and inhibited by ADP, the enzyme showed no response to conventional protein substrates and was essentially independent of pH in the physiological (pH 7 to 8) range. The new protein kinase phosphorylated the mature form of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase and, to a lesser extent, an unidentified 24-kDa polypeptide, both of which were bound to the outer envelope membrane. The results suggest that phosphorylation of cytoplasmically synthesized protein constituents of chloroplasts is involved in their transport through the chloroplast envelope membrane barrier.

  19. Water Dynamics at Protein-Protein Interfaces: Molecular Dynamics Study of Virus-Host Receptor Complexes.

    PubMed

    Dutta, Priyanka; Botlani, Mohsen; Varma, Sameer

    2014-12-26

    The dynamical properties of water at protein-water interfaces are unlike those in the bulk. Here we utilize molecular dynamics simulations to study water dynamics in interstitial regions between two proteins. We consider two natural protein-protein complexes, one in which the Nipah virus G protein binds to cellular ephrin B2 and the other in which the same G protein binds to ephrin B3. While the two complexes are structurally similar, the two ephrins share only a modest sequence identity of ∼50%. X-ray crystallography also suggests that these interfaces are fairly extensive and contain exceptionally large amounts of waters. We find that while the interstitial waters tend to occupy crystallographic sites, almost all waters exhibit residence times of less than hundred picoseconds in the interstitial region. We also find that while the differences in the sequence of the two ephrins result in quantitative differences in the dynamics of interstitial waters, the trends in the shifts with respect to bulk values are similar. Despite the high wetness of the protein-protein interfaces, the dynamics of interstitial waters are considerably slower compared to the bulk-the interstitial waters diffuse an order of magnitude slower and have 2-3 fold longer hydrogen bond lifetimes and 2-1000 fold slower dipole relaxation rates. To understand the role of interstitial waters, we examine how implicit solvent models compare against explicit solvent models in producing ephrin-induced shifts in the G conformational density. Ephrin-induced shifts in the G conformational density are critical to the allosteric activation of another viral protein that mediates fusion. We find that in comparison with the explicit solvent model, the implicit solvent model predicts a more compact G-B2 interface, presumably because of the absence of discrete waters at the G-B2 interface. Simultaneously, we find that the two models yield strikingly different induced changes in the G conformational density, even

  20. Molecular Cloning, mRNA Expression, and Localization of the G-protein Subunit Galphaq in Sheep Testis and Epididymis

    PubMed Central

    Li, Zhen; Lu, Jieli; Sun, Xiaowei; Pang, Quanhai; Zhao, Yiwen

    2016-01-01

    The reproductive function of G-protein subunit Galphaq (GNAQ), a member of the G protein alpha subunit family, has been extensively studied in humans and rats. However, no data is available on its status in ruminants. The objectives of this study were to evaluate the expression pattern of the GNAQ in the testis and epididymis of sheep by polymerase chain reaction (PCR). The mRNA expression levels were detected by real-time fluorescent quantitative PCR, and cellular localization of GNAQ in the testis and epididymis was examined by immunohistochemistry. Additionally, GNAQ protein was qualitatively evaluated via western blot, with the results indicating that similarities between GNAQ mRNA levels from sheep was highly conserved with those observed in Bos taurus and Sus scrofa. Our results also indicated that GNAQ exists in the caput and cauda epididymis of sheep, while GNAQ in the testis and epididymis was localized to Leydig cells, spermatogonial stem cells, spermatocytes, Sertoli cells, spermatid, principal cells, and epididymis interstitial cells. The concentrations of GNAQ mRNA and protein in the caput and cauda epididymis were significantly greater than those observed in the corpus epididymis (p<0.01) and testis (p<0.05). Our results indicated that GNAQ exists at high concentrations in the caput and cauda epididymis of sheep, suggesting that GNAQ may play an important role in gonad development and sperm maturation. PMID:27004818

  1. Immunogenicity Analysis of a Novel Subunit Vaccine Candidate Molecule-Recombinant L7/L12 Ribosomal Protein of Brucella suis.

    PubMed

    Du, Zhi-Qiang; Li, Xin; Wang, Jian-Ying

    2016-08-01

    Brucella was an intracellular parasite, which could infect special livestock and humans. After infected by Brucella, livestock's reproductive system could be affected and destroyed resulting in huge economic losses. More seriously, it could be contagious from livestock to humans. So far, there is no available vaccine which is safe enough for humans. On this point, subunit vaccine has become the new breakthrough of conquering brucellosis. In this study, Brucella rL7/L12-BLS fusion protein was used as an antigen to immunize rabbits to detect the immunogenicity. The results of antibody level testing assay of rabbit antiserum indicated rL7/L12-BLS fusion protein could elicit rabbits to produce high-level IgG. And gamma interferon (IFN-γ) concentrations in rabbit antiserum were obviously up-regulated in both the rL7/L12 group and rL7/L12-BLS group. Besides, the results of quantitative real-time PCR (qRT-PCR) showed the IFN-γ gene's expression levels of both the rL7/L12 group and rL7/L12-BLS group were obviously up-regulated. All these results suggested Brucella L7/L12 protein was an ideal subunit vaccine candidate and possessed good immunogenicity. And Brucella lumazine synthase (BLS) molecule was a favorable transport vector for antigenic protein.

  2. The effect of GABA stimulation on GABAA receptor subunit protein and mRNA expression in rat cultured cerebellar granule cells.

    PubMed Central

    Platt, K. P.; Zwartjes, R. E.; Bristow, D. R.

    1996-01-01

    1. After 8 days in vitro, rat cerebellar granule cells were exposed to 1 mM gamma-aminobutyric acid (GABA) for periods of 1, 2, 4, 6, 8 and 10 days. The effect of the GABA exposure on GABAA receptor alpha 1, alpha 6 and beta 2,3 subunit protein expression and alpha 1 and alpha 6 subunit steady-state mRNA levels, was examined using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. 2. GABA exposure for 2 days decreased alpha 1 (35 +/- 10%, mean +/- s.e.mean), beta 2,3 (21 +/- 9%) and alpha 6 (28 +/- 10%) subunit protein expression compared to control levels. The GABA-mediated reduction in alpha 1 subunit expression after 2 days treatment was abolished in the presence of the GABAA receptor antagonist, Ru 5135 (10 microM). 3. GABA exposure for 8 days increased alpha 1 (26 +/- 10%, mean +/- s.e.mean) and beta 2,3 (56 +/- 23%) subunit protein expression over control levels, whereas alpha 6 subunit protein expression remained below control levels (by 38 +/- 10%). However, after 10 days GABA exposure, alpha 6 subunit protein expression was also increased over control levels by 65 +/- 29% (mean +/- s.e.mean). 4. GABA exposure did not change the alpha 1 or alpha 6 subunit steady-state mRNA levels over and 8 day period, nor did it alter the expression of cyclophilin mRNA over 1-8 days. 5. These results suggest that chronic GABA exposure of rat cerebellar granule cells has a bi-phasic effect on GABAA receptor subunit expression that is independent of changes to mRNA levels. Therefore, the regulation of the GABAA receptor expression by chronic agonist treatment appears to involve post-transcriptional and/or post-translational processes. Images Figure 1 Figure 3 Figure 4 PMID:8968548

  3. Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

    SciTech Connect

    Bhaskar,; Kumari, Neeti; Goyal, Neena

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer The study presents cloning and characterization of TCP1{gamma} gene from L. donovani. Black-Right-Pointing-Pointer TCP1{gamma} is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. Black-Right-Pointing-Pointer LdTCP{gamma} exhibited differential expression in different stages of promastigotes. Black-Right-Pointing-Pointer LdTCP{gamma} co-localized with actin, a cytoskeleton protein. Black-Right-Pointing-Pointer The data suggests that this gene may have a role in differentiation/biogenesis. Black-Right-Pointing-Pointer First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1{gamma}), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1{gamma} of Leishmania donovani (LdTCP1{gamma}), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1{gamma} revealed the presence of all the characteristic features of TCP1{gamma}. However, leishmanial TCP1{gamma} represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1{gamma} exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1{gamma} as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1{gamma} was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1{gamma} with actin suggests

  4. [C825T polymorphism of the GNB3 gene codifying the G-protein beta3-subunit and cardiovascular risk].

    PubMed

    Sartori, Michelangelo; Parotto, Emanuela; Ceolotto, Giulio; Papparella, Italia; Lenzini, Livia; Calò, Lorenzo A; Semplicini, Andrea

    2004-01-01

    Hypertension is a common disorder of multifactorial origin that constitutes a major risk factor for cardiovascular events such as stroke and myocardial infarction. The subunits of the heterotrimeric G proteins are attractive candidate gene products for both susceptibility to essential hypertension and interindividual variation in blood pressure. A polymorphism (825C/T) in exon 10 of the GNB3 gene, that encodes for the beta3 subunit, has recently been described. The 825T allele is associated with alternative splicing of the gene and formation of a truncated but functionally active beta3 subunit. Carriers of the 825T allele appear to have an increased risk for hypertension, obesity, insulin-resistance and left ventricular hypertrophy. Moreover, 825T allele carriers respond with a stronger decrease in blood pressure to therapy with a thiazide diuretic and with clonidine. GNB3 825T allele may be regarded as a potential genetic marker for a better definition of the risk profile of hypertensive subjects, but further studies are needed to precisely define the impact of T allele on the prognosis of such patients.

  5. Broad-Bandwidth Chiral Sum Frequency Generation Spectroscopy for Probing the Kinetics of Proteins at Interfaces.

    PubMed

    Wang, Zhuguang; Fu, Li; Ma, Gang; Yan, Elsa C Y

    2015-10-27

    The kinetics of proteins at interfaces plays an important role in biological functions and inspires solutions to fundamental problems in biomedical sciences and engineering. Nonetheless, due to the lack of surface-specific and structural-sensitive biophysical techniques, it still remains challenging to probe protein kinetics in situ and in real time without the use of spectroscopic labels at interfaces. Broad-bandwidth chiral sum frequency generation (SFG) spectroscopy has been recently developed for protein kinetic studies at interfaces by tracking the chiral vibrational signals of proteins. In this article, we review our recent progress in kinetic studies of proteins at interfaces using broad-bandwidth chiral SFG spectroscopy. We illustrate the use of chiral SFG signals of protein side chains in the C-H stretch region to monitor self-assembly processes of proteins at interfaces. We also present the use of chiral SFG signals from the protein backbone in the N-H stretch region to probe the real-time kinetics of proton exchange between protein and water at interfaces. In addition, we demonstrate the applications of spectral features of chiral SFG that are typical of protein secondary structures in both the amide I and the N-H stretch regions for monitoring the kinetics of aggregation of amyloid proteins at membrane surfaces. These studies exhibit the power of broad-bandwidth chiral SFG to study protein kinetics at interfaces and the promise of this technique in research areas of surface science to address fundamental problems in biomedical and material sciences.

  6. Conservation of coevolving protein interfaces bridges prokaryote-eukaryote homologies in the twilight zone.

    PubMed

    Rodriguez-Rivas, Juan; Marsili, Simone; Juan, David; Valencia, Alfonso

    2016-12-27

    Protein-protein interactions are fundamental for the proper functioning of the cell. As a result, protein interaction surfaces are subject to strong evolutionary constraints. Recent developments have shown that residue coevolution provides accurate predictions of heterodimeric protein interfaces from sequence information. So far these approaches have been limited to the analysis of families of prokaryotic complexes for which large multiple sequence alignments of homologous sequences can be compiled. We explore the hypothesis that coevolution points to structurally conserved contacts at protein-protein interfaces, which can be reliably projected to homologous complexes with distantly related sequences. We introduce a domain-centered protocol to study the interplay between residue coevolution and structural conservation of protein-protein interfaces. We show that sequence-based coevolutionary analysis systematically identifies residue contacts at prokaryotic interfaces that are structurally conserved at the interface of their eukaryotic counterparts. In turn, this allows the prediction of conserved contacts at eukaryotic protein-protein interfaces with high confidence using solely mutational patterns extracted from prokaryotic genomes. Even in the context of high divergence in sequence (the twilight zone), where standard homology modeling of protein complexes is unreliable, our approach provides sequence-based accurate information about specific details of protein interactions at the residue level. Selected examples of the application of prokaryotic coevolutionary analysis to the prediction of eukaryotic interfaces further illustrate the potential of this approach.

  7. Molecular mechanisms of benzodiazepine-induced down-regulation of GABAA receptor alpha 1 subunit protein in rat cerebellar granule cells.

    PubMed Central

    Brown, M. J.; Bristow, D. R.

    1996-01-01

    1. Chronic benzodiazepine treatment of rat cerebellar granule cells induced a transient down-regulation of the gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit protein, that was dose-dependent (1 nM-1 microM) and prevented by the benzodiazepine antagonist flumazenil (1 microM). After 2 days of treatment with 1 microM flunitrazepam the alpha 1 subunit protein was reduced by 41% compared to untreated cells, which returned to, and remained at, control cell levels from 4-12 days of treatment. Chronic flunitrazepam treatment did not significantly alter the GABAA receptor alpha 6 subunit protein over the 2-12 day period. 2. GABA treatment for 2 days down-regulates the alpha 1 subunit protein in a dose-dependent (10 microM-1 mM) manner that was prevented by the selective GABAA receptor antagonist bicuculline (10 microM). At 10 microM and 1 mM GABA the reduction in alpha 1 subunit expression compared to controls was 31% and 66%, respectively. 3. The flunitrazepam-induced decrease in alpha 1 subunit protein is independent of GABA, which suggests that it involves a mechanism distinct from the GABA-dependent action of benzodiazepines on GABAA receptor channel activity. 4. Simultaneous treatment with flunitrazepam and GABA did not produce an additive down-regulation of alpha 1 subunit protein, but produced an effect of the same magnitude as that of flunitrazepam alone. This down-regulation induced by the combination of flunitrazepam and GABA was inhibited by flumazenil (78%), but unaffected by bicuculline. 5. The flunitrazepam-induced down-regulation of alpha 1 subunit protein at 2 days was completely reversed by the protein kinase inhibitor staurosporine (0.3 microM). 6. This study has shown that both flunitrazepam and GABA treatment, via their respective binding sites, caused a reduction in the expression of the GABAA receptor alpha 1 subunit protein; an effect mediated through the same neurochemical mechanism. The results also imply that the benzodiazepine effect

  8. Steady-state levels of G-protein beta-subunit expression are regulated by treatment of cells with bacterial toxins

    SciTech Connect

    Watkins, D.C.; Northup, J.K.; Malbon, C.C.

    1987-05-01

    Cultures of 3T3-L1 cells were incubated with either 10 ng/ml cholera toxin or 10 ng/ml pertussis toxin from 4 days prior to the initiation of differentiation and throughout the subsequent incubation. Toxin concentrations were sufficient to completely prevent the labelling of alpha-subunits with (/sup 32/P)NAD/sup +/ and pertussis toxin and to prevent by more than 90% the labelling with (/sup 32/P)NAD/sup +/ and cholera toxin in membranes prepared from these cells. Neither toxin prevented the differentiation to the adipocyte phenotype. Neither toxin prevented the increases in the relative amounts of G-proteins which occur upon differentiation. Both toxins dramatically decreased the amount of beta-subunits. As measured by quantitative immunoblotting with antisera specific for both the 35 kDa and 36 kDa beta-subunits, levels of beta-subunit were decreased by more than 50% of steady-state level of control cells. Thus, bacterial toxins which modifies G-protein alpha-subunits are capable of modulating the levels of beta-subunits in vivo. The basis for the regulation of G-protein subunit expression by bacterial toxins is under study.

  9. Functional analysis of the glycogen binding subunit CG9238/Gbs-70E of protein phosphatase 1 in Drosophila melanogaster.

    PubMed

    Kerekes, Éva; Kókai, Endre; Páldy, Ferenc Sándor; Dombrádi, Viktor

    2014-06-01

    The product of the CG9238 gene that we termed glycogen binding subunit 70E (Gbs-70E) was characterized by biochemical and molecular genetics methods. The interaction between Gbs-70E and all catalytic subunits of protein phosphatase 1 (Pp1-87B, Pp1-9C, Pp1-96A and Pp1-13C) of Drosophila melanogaster was confirmed by pairwise yeast two-hybrid tests, co-immunoprecipitation and pull down experiments. The binding of Gbs-70E to glycogen was demonstrated by sedimentation analysis. With RT-PCR we found that the mRNAs coding for the longer Gbs-70E PB/PC protein were expressed in all developmental stages of the fruit flies while the mRNA for the shorter Gbs-70E PA was restricted to the eggs and the ovaries of the adult females. The development specific expression of the shorter splice variant was not conserved in different Drosophila species. The expression level of the gene was manipulated by P-element insertions and gene deletion to analyze the functions of the gene product. A small or moderate reduction in the gene expression resulted in no significant changes, however, a deletion mutant expressing very low level of the transcript lived shorter and exhibited reduced glycogen content in the imagos. In addition, the gene deletion decreased the fertility of the fruit flies. Our results prove that Gbs-70E functions as the glycogen binding subunit of protein phosphatase 1 that regulates glycogen content and plays a role in the development of eggs in D. melanogaster.

  10. EPR Studies of Functionally Active, Nitroxide Spin-Labeled Peptide Analogs of the C-terminus of a G-Protein Alpha Subunit

    PubMed Central

    Van Eps, Ned; Anderson, Lori L.; Kisselev, Oleg G.; Baranski, Thomas J.; Hubbell, Wayne L.; Marshall, Garland R.

    2010-01-01

    The C-terminal tail of the transducin alpha subunit, Gtα(340–350), is known to bind and stabilize the active conformation of rhodopsin upon photoactivation (R*). Five spin-labeled analogs of Gtα(340–350) demonstrated native-like activity in their ability to bind and stabilize R*. The spin label 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) was employed at interior sites within the peptide, whereas a Proxyl (3-carboxyl-2,2,5,5-tetramethyl-pyrrolidinyloxy) spin label was employed at the amino terminus of the peptide. Upon binding to R*, the electron paramagnetic resonance spectrum of TOAC343-Gtα(340–350) revealed greater immobilization of the nitroxide when compared to that of the N-terminal modified Proxyl-Gtα(340–350) analog. A double-labeled Proxyl/TOAC348-Gtα(340–350) was examined by DEER spectroscopy to determine the distribution of distances between the two nitroxides in the peptides when in solution and when bound to R*. TOAC and Proxyl spin labels in this GPCR-G-protein α-peptide system provide unique biophysical probes that can be used to explore the structure and conformational changes at the rhodopsin-G-protein interface. PMID:20695526

  11. Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits

    PubMed Central

    Shi, Jiong; Friedman, David B.

    2013-01-01

    The host restriction factors TRIM5α and TRIMCyp potently inhibit retrovirus infection by binding to the incoming retrovirus capsid. TRIM5 proteins are dimeric, and their association with the viral capsid appears to be enhanced by avidity effects owing to formation of higher-order oligomeric complexes. We examined the stoichiometric requirement for TRIM5 functional recognition by quantifying the efficiencies of restriction of HIV-1 and murine leukemia virus (MLV) particles containing various proportions of restriction-sensitive and -insensitive CA subunits. Both TRIMCyp and TRIM5α inhibited infection of retrovirus particles containing as little as 25% of the restriction-sensitive CA protein. Accordingly, we also observed efficient binding of TRIMCyp in vitro to capsid assemblies containing as little as one-fourth wild-type CA protein. Paradoxically, the ability of HIV-1 particles to abrogate TRIMCyp restriction in trans was more strongly dependent on the fraction of wild-type CA than was restriction of infection. Collectively, our results indicate that TRIM5 restriction factors bind to retroviral capsids in a highly cooperative manner and suggest that TRIM5 can engage a capsid lattice containing a minimum of three or fewer recognizable subunits per hexamer. Our study supports a model in which localized binding of TRIM5 to the viral capsid nucleates rapid polymerization of a TRIM5 lattice on the capsid surface. PMID:23785198

  12. The F13 residue is critical for interaction among the coat protein subunits of papaya mosaic virus.

    PubMed

    Laliberté Gagné, M E; Lecours, K; Gagné, S; Leclerc, D

    2008-04-01

    Papaya mosaic virus (PapMV) coat protein (CP) in Escherichia coli was previously showed to self-assemble in nucleocapsid-like particles (NLPs) that were similar in shape and appearance to the native virus. We have also shown that a truncated CP missing the N-terminal 26 amino acids is monomeric and loses its ability to bind RNA. It is likely that the N-terminus of the CP is important for the interaction between the subunits in self-assembly into NLPs. In this work, through deletion and mutation analysis, we have shown that the deletion of 13 amino acids is sufficient to generate the monomeric form of the CP. Furthermore, we have shown that residue F13 is critical for self-assembly of the CP subunits into NLPs. The replacement of F13 with hydrophobic residues (L or Y) generated mutated forms of the CP that were able to self-assemble into NLPs. However, the replacement of F13 by A, G, R, E or S was detrimental to the self-assembly of the protein into NLPs. We concluded that a hydrophobic interaction at the N-terminus is important to ensure self-assembly of the protein into NLPs. We also discuss the importance of F13 for assembly of other members of the potexvirus family.

  13. Protein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase.

    PubMed

    Cardinale, Daniela; Guaitoli, Giambattista; Tondi, Donatella; Luciani, Rosaria; Henrich, Stefan; Salo-Ahen, Outi M H; Ferrari, Stefania; Marverti, Gaetano; Guerrieri, Davide; Ligabue, Alessio; Frassineti, Chiara; Pozzi, Cecilia; Mangani, Stefano; Fessas, Dimitrios; Guerrini, Remo; Ponterini, Glauco; Wade, Rebecca C; Costi, M Paola

    2011-08-23

    Human thymidylate synthase is a homodimeric enzyme that plays a key role in DNA synthesis and is a target for several clinically important anticancer drugs that bind to its active site. We have designed peptides to specifically target its dimer interface. Here we show through X-ray diffraction, spectroscopic, kinetic, and calorimetric evidence that the peptides do indeed bind at the interface of the dimeric protein and stabilize its di-inactive form. The "LR" peptide binds at a previously unknown binding site and shows a previously undescribed mechanism for the allosteric inhibition of a homodimeric enzyme. It inhibits the intracellular enzyme in ovarian cancer cells and reduces cellular growth at low micromolar concentrations in both cisplatin-sensitive and -resistant cells without causing protein overexpression. This peptide demonstrates the potential of allosteric inhibition of hTS for overcoming platinum drug resistance in ovarian cancer.

  14. The influence of interfaces on properties of thin-film inorganic structural isomers containing SnSe—NbSe₂ Subunits

    SciTech Connect

    Alemayehu, Matti B.; Falmbigl, Matthias; Ta, Kim; Johnson, David C.

    2016-06-15

    Inorganic isomers ([SnSe]1+δ)m(NbSe₂)n([SnSe]1+δ)p(NbSe₂)q([SnSe]1+δ)r(NbSe₂)s where m, n, p, q, r, and s are integers and m + p + r = n + q + s = 4 were prepared using the modulated elemental reactant technique. This series of all six possible isomers provides an opportunity to study the influence of interface density on properties while maintaining the same unit cell size and composition. As expected, all six compounds were observed to have the same atomic compositions and an almost c-axis lattice parameter of ≈4.90 (5) nm, with a slight trend in the c-axis lattice parameter correlated with the different number of interfaces in the isomers: two, four and six. The structures of the constituents in the ab-plane were independent of one another, confirming the nonepitaxial relationship between them. The temperature dependent electrical resistivities revealed metallic behavior for all the six compounds. Surprisingly, the electrical resistivity at room temperature decreases with increasing number of interfaces. Hall measurements suggest this results from changes in carrier concentration, which increases with increasing thickness of the thickest SnSe block in the isomer. Carrier mobility scales with the thickness of the thickest NbSe₂ block due to increased interfacial scattering as the NbSe₂ blocks become thinner. The observed behavior suggests that the two constituents serve different purposes with respect to electrical transport. SnSe acts as a charge donor and NbSe₂ acts as the charge transport layer. This separation of function suggests that such heterostructures can be designed to optimize performance through choice of constituent, layer thickness, and layer sequence. A simplistic model, which predicts the properties of the complex isomers from a weighted sum of the

  15. A small novel A-kinase anchoring protein (AKAP) that localizes specifically protein kinase A-regulatory subunit I (PKA-RI) to the plasma membrane.

    PubMed

    Burgers, Pepijn P; Ma, Yuliang; Margarucci, Luigi; Mackey, Mason; van der Heyden, Marcel A G; Ellisman, Mark; Scholten, Arjen; Taylor, Susan S; Heck, Albert J R

    2012-12-21

    Protein kinase A-anchoring proteins (AKAPs) provide spatio-temporal specificity for the omnipotent cAMP-dependent protein kinase (PKA) via high affinity interactions with PKA regulatory subunits (PKA-RI, RII). Many PKA-RII-AKAP complexes are heavily tethered to cellular substructures, whereas PKA-RI-AKAP complexes have remained largely undiscovered. Here, using a cAMP affinity-based chemical proteomics strategy in human heart and platelets, we uncovered a novel, ubiquitously expressed AKAP, termed small membrane (sm)AKAP due to its specific localization at the plasma membrane via potential myristoylation/palmitoylation anchors. In vitro binding studies revealed specificity of smAKAP for PKA-RI (K(d) = 7 nM) over PKA-RII (K(d) = 53 nM) subunits, co-expression of smAKAP with the four PKA R subunits revealed an even more exclusive specificity of smAKAP for PKA-RIα/β in the cellular context. Applying the singlet oxygen-generating electron microscopy probe miniSOG indicated that smAKAP is tethered to the plasma membrane and is particularly dense at cell-cell junctions and within filopodia. Our preliminary functional characterization of smAKAP provides evidence that, like PKA-RII, PKA-RI can be tightly tethered by a novel repertoire of AKAPs, providing a new perspective on spatio-temporal control of cAMP signaling.

  16. Secretory proteins are delivered to the septin-organized penetration interface during root infection by Verticillium dahliae

    PubMed Central

    Zhou, Ting-Ting; Zhao, Yun-Long

    2017-01-01

    Successful infection of the host requires secretion of effector proteins to evade or suppress plant immunity. Secretion of effectors in root-infecting fungal pathogens, however, remains unexplored. We previously reported that Verticillium dahliae, a root-infecting phytopathogenic fungus, develops a penetration peg from a hyphopodium to infect cotton roots. In this study, we report that a septin ring, requiring VdSep5, partitions the hyphopodium and the invasive hypha and form the specialized fungus-host interface. The mutant strain, VdΔnoxb, in which NADPH oxidase B (VdNoxB) is deleted, impaired formation of the septin ring at the hyphal neck, indicating that NADPH oxidases regulate septin ring organization. Using GFP tagging and live cell imaging, we observed that several signal peptide containing secreted proteins showed ring signal accumulation/secretion at the penetration interface surrounding the hyphal neck. Targeted mutation for VdSep5 reduced the delivery rate of secretory proteins to the penetration interface. Blocking the secretory pathway by disrupting the vesicular trafficking factors, VdSec22 and VdSyn8, or the exocyst subunit, VdExo70, also arrested delivery of the secreted proteins inside the hyphopodium. Reduced virulence was observed when cotton roots were infected with VdΔsep5, VdΔsec22, VdΔsyn8 and VdΔexo70 mutants compared to infection with the isogenic wild-type V592. Taken together, our data demonstrate that the hyphal neck is an important site for protein secretion during plant root infection, and that the multiple secretory routes are involved in the secretion. PMID:28282450

  17. Identification of a GTP-binding protein. cap alpha. subunit that lacks an apparent ADP-ribosylation site for pertussis toxin

    SciTech Connect

    Fong, H.K.W.; Yoshimoto, K.K.; Eversole-Cire, P.; Simon, M.I.

    1988-05-01

    Recent molecular cloning of cDNA for the ..cap alpha.. subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called T/sub r/ and T/sub c/, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, the authors have identified a G-protein ..cap alpha.. subunit, which they refer to as G/sub z/..cap alpha.., by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine T/sub r/ ..cap alpha..-subunit cDNA. The deduced amino acid sequence of G/sub z/..cap alpha.. is 41-67% identical with those of other known G-protein ..cap alpha.. subunits. However, the 355-residue G/sub z/..cap alpha.. lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein ..cap alpha.. subunits. They suggest that G/sub z/..cap alpha.., which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems.

  18. Transient protein-protein interface prediction: datasets, features, algorithms, and the RAD-T predictor

    PubMed Central

    2014-01-01

    Background Transient protein-protein interactions (PPIs), which underly most biological processes, are a prime target for therapeutic development. Immense progress has been made towards computational prediction of PPIs using methods such as protein docking and sequence analysis. However, docking generally requires high resolution structures of both of the binding partners and sequence analysis requires that a significant number of recurrent patterns exist for the identification of a potential binding site. Researchers have turned to machine learning to overcome some of the other methods’ restrictions by generalising interface sites with sets of descriptive features. Best practices for dataset generation, features, and learning algorithms have not yet been identified or agreed upon, and an analysis of the overall efficacy of machine learning based PPI predictors is due, in order to highlight potential areas for improvement. Results The presence of unknown interaction sites as a result of limited knowledge about protein interactions in the testing set dramatically reduces prediction accuracy. Greater accuracy in labelling the data by enforcing higher interface site rates per domain resulted in an average 44% improvement across multiple machine learning algorithms. A set of 10 biologically unrelated proteins that were consistently predicted on with high accuracy emerged through our analysis. We identify seven features with the most predictive power over multiple datasets and machine learning algorithms. Through our analysis, we created a new predictor, RAD-T, that outperforms existing non-structurally specializing machine learning protein interface predictors, with an average 59% increase in MCC score on a dataset with a high number of interactions. Conclusion Current methods of evaluating machine-learning based PPI predictors tend to undervalue their performance, which may be artificially decreased by the presence of un-identified interaction sites. Changes to

  19. Structural and biochemical characterization of human PR70 in isolation and in complex with the scaffolding subunit of protein phosphatase 2A.

    PubMed

    Dovega, Rebecca; Tsutakawa, Susan; Quistgaard, Esben M; Anandapadamanaban, Madhanagopal; Löw, Christian; Nordlund, Pär

    2014-01-01

    Protein Phosphatase 2A (PP2A) is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A) subunit, a catalytic (C) subunit and various regulatory (B) subunits. Here we report a 2.0 Å crystal structure of the free B''/PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B''/PR70 reveals a two domain elongated structure with two Ca2+ binding EF-hands. Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca2+ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca2+. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B''/PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B'' containing holoenzymes.

  20. Structural and Biochemical Characterization of Human PR70 in Isolation and in Complex with the Scaffolding Subunit of Protein Phosphatase 2A

    PubMed Central

    Dovega, Rebecca; Tsutakawa, Susan; Quistgaard, Esben M.; Anandapadamanaban, Madhanagopal; Löw, Christian; Nordlund, Pär

    2014-01-01

    Protein Phosphatase 2A (PP2A) is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A) subunit, a catalytic (C) subunit and various regulatory (B) subunits. Here we report a 2.0 Å crystal structure of the free B’’/PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B’’/PR70 reveals a two domain elongated structure with two Ca2+ binding EF-hands. Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca2+ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca2+. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B’’/PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B’’ containing holoenzymes. PMID:25007185

  1. The recombinant globular head domain of the measles virus hemagglutinin protein as a subunit vaccine against measles.

    PubMed

    Lobanova, Liubov M; Eng, Nelson F; Satkunarajah, Malathy; Mutwiri, George K; Rini, James M; Zakhartchouk, Alexander N

    2012-04-26

    Despite the availability of live attenuated measles virus (MV) vaccines, a large number of measles-associated deaths occur among infants in developing countries. The development of a measles subunit vaccine may circumvent the limitations associated with the current live attenuated vaccines and eventually contribute to global measles eradication. Therefore, the goal of this study was to test the feasibility of producing the recombinant globular head domain of the MV hemagglutinin (H) protein by stably transfected human cells and to examine the ability of this recombinant protein to elicit MV-specific immune responses. The recombinant protein was purified from the culture supernatant of stably transfected HEK293T cells secreting a tagged version of the protein. Two subcutaneous immunizations with the purified recombinant protein alone resulted in the production of MV-specific serum IgG and neutralizing antibodies in mice. Formulation of the protein with adjuvants (polyphosphazene or alum) further enhanced the humoral immune response and in addition resulted in the induction of cell-mediated immunity as measured by the production of MV H-specific interferon gamma (IFN-γ) and interleukin 5 (IL-5) by in vitro re-stimulated splenocytes. Furthermore, the inclusion of polyphosphazene into the vaccine formulation induced a mixed Th1/Th2-type immune response. In addition, the purified recombinant protein retained its immunogenicity even after storage at 37°C for 2 weeks.

  2. Structural Determination of Biomolecular Interfaces by Nuclear Magnetic Resonance of Proteins with Reduced Proton Density

    PubMed Central

    Ferrage, Fabien; Dutta, Kaushik; Shekhtman, Alexander; Cowburn, David

    2013-01-01

    Protein interactions are important for understanding many molecular mechanisms underlying cellular processes. So far, interfaces between interacting proteins have been characterized by NMR spectroscopy mostly by using chemical shift perturbations and cross-saturation via intermolecular cross-relaxation. Although powerful, these techniques cannot provide unambiguous estimates of intermolecular distances between interacting proteins. Here, we present an alternative approach, called REDSPRINT (REDduced/Standard PRoton density INTerface identification), to map protein interfaces with greater accuracy by using multiple NMR probes. Our approach is based on monitoring the cross-relaxation from a source protein (or from an arbitrary ligand that need not be a protein) with high proton density to a target protein (or other biomolecule) with low proton density using isotope-filtered nuclear Overhauser spectroscopy (NOESY). This methodology uses different isotropic labeling for the source and target proteins to identify the source-target interface and also determine the proton density of the source protein at the interface for protein-protein or protein-ligand docking. The utility of this technique, including a method for direct determination of the protein surface, is demonstrated for two different protein-protein complexes. PMID:20372977

  3. Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

    PubMed Central

    Ocampo, J.; McCormack, B.; Navarro, E.; Moreno, S.; Garre, V.

    2012-01-01

    The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway. PMID:22635921

  4. Developmental regulation of G protein-gated inwardly-rectifying K+ (GIRK/KIR3) channel subunits in the brain

    PubMed Central

    Fernández-Alacid, Laura; Watanabe, Masahiko; Molnár, Elek; Wickman, Kevin; Luján, Rafael

    2013-01-01

    G protein-gated inwardly-rectifying K+ (GIRK/family 3 of inwardly-rectifying K+) channels are coupled to neurotransmitter action and can play important roles in modulating neuronal excitability. We investigated the temporal and spatial expression of GIRK1, GIRK2 and GIRK3 subunits in the developing and adult rodent brain using biochemical, immunohistochemical and immunoelectron microscopic techniques. At all ages analysed, the overall distribution patterns of GIRK1-3 were very similar, with high expression levels in the neocortex, cerebellum, hippocampus and thalamus. Focusing on the hippocampus, histoblotting and immunohistochemistry showed that GIRK1-3 protein levels increased with age, and this was accompanied by a shift in the subcellular localization of the subunits. Early in development (postnatal day 5), GIRK subunits were predominantly localized to the endoplasmic reticulum in the pyramidal cells, but by postnatal day 60 they were mostly found along the plasma membrane. During development, GIRK1 and GIRK2 were found primarily at postsynaptic sites, whereas GIRK3 was predominantly detected at presynaptic sites. In addition, GIRK1 and GIRK2 expression on the spine plasma membrane showed identical proximal-to-distal gradients that differed from GIRK3 distribution. Furthermore, although GIRK1 was never found within the postsynaptic density (PSD), the level of GIRK2 in the PSD progressively increased and GIRK3 did not change in the PSD during development. Together, these findings shed new light on the developmental regulation and subcellular diversity of neuronal GIRK channels, and support the contention that distinct subpopulations of GIRK channels exert separable influences on neuronal excitability. The ability to selectively target specific subpopulations of GIRK channels may prove effective in the treatment of disorders of excitability. PMID:22098295

  5. Tumor necrosis factor receptor-associated protein 1 improves hypoxia-impaired energy production in cardiomyocytes through increasing activity of cytochrome c oxidase subunit II.

    PubMed

    Xiang, Fei; Ma, Si-Yuan; Zhang, Dong-Xia; Zhang, Qiong; Huang, Yue-Sheng

    2016-10-01

    Tumor necrosis factor receptor-associated protein 1 protects cardiomyocytes against hypoxia, but the underlying mechanisms are not completely understood. In the present study, we used gain- and loss-of-function approaches to explore the effects of tumor necrosis factor receptor-associated protein 1 and cytochrome c oxidase subunit II on energy production in hypoxic cardiomyocytes. Hypoxia repressed ATP production in cultured cardiomyocytes, whereas overexpression of tumor necrosis factor receptor-associated protein 1 significantly improved ATP production. Conversely, knockdown of tumor necrosis factor receptor-associated protein 1 facilitated the hypoxia-induced decrease in ATP synthesis. Further investigation revealed that tumor necrosis factor receptor-associated protein 1 induced the expression and activity of cytochrome c oxidase subunit II, a component of cytochrome c oxidase that is important in mitochondrial respiratory chain function. Moreover, lentiviral-mediated overexpression of cytochrome c oxidase subunit II antagonized the decrease in ATP synthesis caused by knockdown of tumor necrosis factor receptor-associated protein 1, whereas knockdown of cytochrome c oxidase subunit II attenuated the increase in ATP synthesis caused by overexpression of tumor necrosis factor receptor-associated protein 1. In addition, inhibition of cytochrome c oxidase subunit II by a specific inhibitor sodium azide suppressed the ATP sy nthesis induced by overexpressed tumor necrosis factor receptor-associated protein 1. Hence, tumor necrosis factor receptor-associated protein 1 protects cardiomyocytes from hypoxia at least partly via potentiation of energy generation, and cytochrome c oxidase subunit II is one of the downstream effectors that mediates the tumor necrosis factor receptor-associated protein 1-mediated energy generation program.

  6. Immunoproteomic analysis of Brucella melitensis and identification of a new immunogenic candidate protein for the development of brucellosis subunit vaccine.

    PubMed

    Yang, Yanling; Wang, Lin; Yin, Jigang; Wang, Xinglong; Cheng, Shipeng; Lang, Xulong; Wang, Xiuran; Qu, Hailong; Sun, Chunhui; Wang, Jinglong; Zhang, Rui

    2011-10-01

    In order to screen immunogenic candidate antigens for the development of a brucellosis subunit vaccine, an immunoproteomic assay was used to identify immunogenic proteins from Brucella melitensis 16 M soluble proteins. In this study, a total of 56 immunodominant proteins were identified from the two-dimensional electrophoresis immunoblot profiles by liquid chromatography tandem mass spectrometry (LC-MS/MS). Two proteins of interest, riboflavin synthase alpha chain (RS-α) and Loraine synthase (LS-2), which are both involved in riboflavin synthesis, were detected by two-dimensional immunoblots using antisera obtained from Brucella-infected human and goats. LS-2, however, is an already well-known vaccine candidate. Therefore, we focussed our studies on the novel vaccine candidate RS-α. B. melitensis RS-α and LS-2 were then expressed in Escherichia coli as fusion proteins with His tag. The humoral and cellular immune responses to the recombinant (r)RS-α was characterized. In response to in vitro stimulation by rRS-α, splenocytes from mice vaccinated with rRS-α were able to produce γ-interferon (IFN-γ) and interleukin (IL)-2 but not interleukin (IL)-4 and interleukin (IL)-10. Furthermore, rRS-α or rLS-2-vaccinated mice were partially protected against B. melitensis infection. Our results suggested that we have developed a high-throughout, accurate, rapid and highly efficient method for the identification of candidate antigens by a combination of immunoproteomics with immunisation and bacterial challenge and rRs-α could be a useful candidate for the development of subunit vaccines against B. melitensis.

  7. Crystallization of the glycogen-binding domain of the AMP-activated protein kinase β subunit and preliminary X-ray analysis

    PubMed Central

    Polekhina, Galina; Feil, Susanne C.; Gupta, Abhilasha; O’Donnell, Paul; Stapleton, David; Parker, Michael W.

    2005-01-01

    AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolism in response to energy demand and supply by adjusting the ATP-generating and ATP-consuming pathways. AMPK potentially plays a critical role in diabetes and obesity as it is known to be activated by metforin and rosiglitazone, drugs used for the treatment of type II diabetes. AMPK is a heterotrimer composed of a catalytic α subunit and two regulatory subunits, β and γ. Mutations in the γ subunit are known to cause glycogen accumulation, leading to cardiac arrhythmias. Recently, a functional glycogen-binding domain (GBD) has been identified in the β subunit. Here, the crystallization of GBD in the presence of β-cyclodextrin is reported together with preliminary X-ray data analysis allowing the determination of the structure by single isomorphous replacement and threefold averaging using in-house X-ray data collected from a selenomethionine-substituted protein. PMID:16508085

  8. Substrate-Induced Unfolding of Protein Disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from Its Holotoxin

    PubMed Central

    Taylor, Michael; Burress, Helen; Banerjee, Tuhina; Ray, Supriyo; Curtis, David; Tatulian, Suren A.; Teter, Ken

    2014-01-01

    To generate a cytopathic effect, the catalytic A1 subunit of cholera toxin (CT) must be separated from the rest of the toxin. Protein disulfide isomerase (PDI) is thought to mediate CT disassembly by acting as a redox-driven chaperone that actively unfolds the CTA1 subunit. Here, we show that PDI itself unfolds upon contact with CTA1. The substrate-induced unfolding of PDI provides a novel molecular mechanism for holotoxin disassembly: we postulate the expanded hydrodynamic radius of unfolded PDI acts as a wedge to dislodge reduced CTA1 from its holotoxin. The oxidoreductase activity of PDI was not required for CT disassembly, but CTA1 displacement did not occur when PDI was locked in a folded conformation or when its substrate-induced unfolding was blocked due to the loss of chaperone function. Two other oxidoreductases (ERp57 and ERp72) did not unfold in the presence of CTA1 and did not displace reduced CTA1 from its holotoxin. Our data establish a new functional property of PDI that may be linked to its role as a chaperone that prevents protein aggregation. PMID:24516389

  9. Quantitative Phosphoproteomics Reveals Novel Phosphorylation Events in Insulin Signaling Regulated by Protein Phosphatase 1 Regulatory Subunit 12A

    PubMed Central

    Zhang, Xiangmin; Ma, Danjun; Caruso, Michael; Lewis, Monique; Qi, Yue; Yi, Zhengping

    2014-01-01

    Serine/threonine protein phosphatase 1 regulatory subunit 12A (PPP1R12A) modulates the activity and specificity of the catalytic subunit of protein phosphatase 1, regulating various cellular processes via dephosphorylation. Nonetheless, little is known about phosphorylation events controlled by PPP1R12A in skeletal muscle insulin signaling. Here, we used quantitative phosphoproteomics to generate a global picture of phosphorylation events regulated by PPP1R12A in a L6 skeletal muscle cell line, which were engineered for inducible PPP1R12A knockdown. Phosphoproteomics revealed 3876 phosphorylation sites (620 were novel) in these cells. Furthermore, PPP1R12A knockdown resulted in increased overall phosphorylation in L6 cells at the basal condition, and changed phosphorylation levels for 698 sites (assigned to 295 phosphoproteins) at the basal and/or insulin-stimulated conditions. Pathway analysis on the 295 phosphoproteins revealed multiple significantly enriched pathways related to insulin signaling, such as mTOR signaling and RhoA signaling. Moreover, phosphorylation levels for numerous regulatory sites in these pathways were significantly changed due to PPP1R12A knockdown. These results indicate that PPP1R12A indeed plays a role in skeletal muscle insulin signaling, providing novel insights into the biology of insulin action. This new information may facilitate the design of experiments to better understand mechanisms underlying skeletal muscle insulin resistance and type 2 diabetes. PMID:24972320

  10. Activation of phospholipase C by the alpha subunits of the Gq and G11 proteins in transfected Cos-7 cells.

    PubMed

    Wu, D Q; Lee, C H; Rhee, S G; Simon, M I

    1992-01-25

    High efficiency transient transfection was used to introduce cDNA corresponding to various G protein alpha subunits into Cos-7 cells. The proteins that were subsequently synthesized were detected with specific G protein alpha subunit antipeptide antiserum and were localized in the membrane fraction of the cell. Cells that were prelabeled with the [3H]inositol and transfected with G alpha q and G alpha 11 cDNA showed marked increases in formation of [3H]inositol phosphates after stimulation with aluminum fluoride. Co-transfection with cDNAs corresponding to phosphoinositide specific phospholipase C beta 1 (PI-PLC beta 1) and to G alpha q or G alpha 11 resulted in even higher levels of inositol phosphate formation. The introduction of mutations that convert residue glutamine 209 to leucine in G alpha q and G alpha 11 resulted in persistent activation of PI-PLC and high steady state levels of inositol phosphates. On the other hand, transfection with a variety of other G alpha subunit cDNAs, i.e. G alpha Z, G alpha OA, G alpha OB, transducin, and the glutamine 205 to leucine mutants of G alpha Z and of G alpha OA did not increase inositol phosphate formation. To further test the specificity of G protein activation of PI-PLC, a cell-free system was prepared by using washed membranes of transiently transfected cells and purified PI-PLC beta 1. Membranes derived from G alpha q and G alpha 11, but not G alpha OA transfected cells, showed guanosine 5-O-thiotriphosphate (GTP gamma S)-stimulated PIP2 hydrolysis. The activity seen in the system reconstituted with membranes derived from G alpha 11-transfected cells was blocked by preincubation with specific G alpha 11 antipeptide antibodies. All of these results are consistent with the conclusion that G alpha q and G alpha 11 cDNA encode proteins that in the presence of GTP gamma S specifically activate PI-PLC.

  11. Nonclathrin coat protein gamma, a subunit of coatomer, binds to the cytoplasmic dilysine motif of membrane proteins of the early secretory pathway.

    PubMed Central

    Harter, C; Pavel, J; Coccia, F; Draken, E; Wegehingel, S; Tschochner, H; Wieland, F

    1996-01-01

    Coatomer, a cytosolic heterooligomeric protein complex that consists of seven subunits [alpha-, beta-, beta'-, gamma-, delta-, epsilon-, and zeta-COP (nonclathrin coat protein)], has been shown to interact with dilysine motifs typically found in the cytoplasmic domains of various endoplasmic-reticulum-resident membrane proteins [Cosson, P. & Letourneur, F. (1994) Science 263, 1629-1631]. We have used a photo-cross-linking approach to identify the site of coatomer that is involved in binding to the dilysine motifs. An octapeptide corresponding to the C-terminal tail of Wbp1p, a component of the yeast N-oligosaccharyltransferase complex, has been synthesized with a photoreactive phenylalanine at position -5 and was radioactively labeled with [125I]iodine at a tyrosine residue introduced at the N terminus of the peptide. Photolysis of isolated coatomer in the presence of this peptide and immunoprecipitation of coatomer from photo-cross-linked cell lysates reveal that gamma-COP is the predominantly labeled protein. From these results, we conclude that coatomer is able to bind to the cytoplasmic dilysine motifs of membrane proteins of the early secretory pathway via its gamma-COP subunit, whose complete cDNA-derived amino acid sequence is also presented. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8700856

  12. Protein Interaction between Ameloblastin and Proteasome Subunit α Type 3 Can Facilitate Redistribution of Ameloblastin Domains within Forming Enamel.

    PubMed

    Geng, Shuhui; White, Shane N; Paine, Michael L; Snead, Malcolm L

    2015-08-21

    Enamel is a bioceramic tissue composed of thousands of hydroxyapatite crystallites aligned in parallel within boundaries fabricated by a single ameloblast cell. Enamel is the hardest tissue in the vertebrate body; however, it starts development as a self-organizing assembly of matrix proteins that control crystallite habit. Here, we examine ameloblastin, a protein that is initially distributed uniformly across the cell boundary but redistributes to the lateral margins of the extracellular matrix following secretion thus producing cell-defined boundaries within the matrix and the mineral phase. The yeast two-hybrid assay identified that proteasome subunit α type 3 (Psma3) interacts with ameloblastin. Confocal microscopy confirmed Psma3 co-distribution with ameloblastin at the ameloblast secretory end piece. Co-immunoprecipitation assay of mouse ameloblast cell lysates with either ameloblastin or Psma3 antibody identified each reciprocal protein partner. Protein engineering demonstrated that only the ameloblastin C terminus interacts with Psma3. We show that 20S proteasome digestion of ameloblastin in vitro generates an N-terminal cleavage fragment consistent with the in vivo pattern of ameloblastin distribution. These findings suggest a novel pathway participating in control of protein distribution within the extracellular space that serves to regulate the protein-mineral interactions essential to biomineralization.

  13. Protein kinase A type II-α regulatory subunit regulates the response of prostate cancer cells to taxane treatment

    PubMed Central

    Zynda, Evan R; Matveev, Vitaliy; Makhanov, Michael; Chenchik, Alexander; Kandel, Eugene S

    2014-01-01

    In the last decade taxane-based therapy has emerged as a standard of care for hormone-refractory prostate cancer. Nevertheless, a significant fraction of tumors show no appreciable response to the treatment, while the others develop resistance and recur. Despite years of intense research, the mechanisms of taxane resistance in prostate cancer and other malignancies are poorly understood and remain a topic of intense investigation. We have used improved mutagenesis via random insertion of a strong promoter to search for events, which enable survival of prostate cancer cells after Taxol exposure. High-throughput mapping of the integration sites pointed to the PRKAR2A gene, which codes for a type II-α regulatory subunit of protein kinase A, as a candidate modulator of drug response. Both full-length and N-terminally truncated forms of the PRKAR2A gene product markedly increased survival of prostate cancer cells lines treated with Taxol and Taxotere. Suppression of protein kinase A enzymatic activity is the likely mechanism of action of the overexpressed proteins. Accordingly, protein kinase A inhibitor PKI (6–22) amide reduced toxicity of Taxol to prostate cancer cells. Our findings support the role of protein kinase A and its constituent proteins in cell response to chemotherapy. PMID:25485509

  14. Fate of secretory proteins trapped in oocytes of Xenopus laevis by disruption of the cytoskeleton or by imbalanced subunit synthesis

    PubMed Central

    1981-01-01

    The effects of imbalanced subunit synthesis, temperature, colchicine, and cytochalasin on the secretion from Xenopus laevis oocytes of a variety of avian and mammalian proteins were investigated; these proteins were encoded by microinjected messenger RNA. Cytochalasin and colchicine together severely reduced secretion in a temperature- independent manner, the exact reduction varying among the different proteins. In contrast cytochalasin alone had no effect, whereas colchicine alone caused a smaller, temperature-dependent reduction. The synthesis and subcellular compartmentation of these proteins were unaffected by the drug treatments; however, the proteins did not accumulate in the drug-treated oocytes but were degraded. The rate of degradation of each protein was similar to its rate of exocytosis from untreated oocytes. A similar result was obtained without recourse to drugs by studying the fate of immunoglobulin light chains trapped in oocytes by a deficiency in heavy chain synthesis. These results are discussed in terms of the disruptive effects, as revealed by electron microscopy, of the drug treatments on the cytoskeleton of the oocyte. PMID:6173386

  15. DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis.

    PubMed

    Lira, C B B; Gui, K E; Perez, A M; da Silveira, R C V; Gava, L M; Ramos, C H I; Cano, M I N

    2009-02-01

    Replication protein A (RPA) is a single-stranded DNA-binding protein that has been implicated in DNA metabolism and telomere maintenance. Subunit 1 of RPA from Leishmania amazonensis (LaRPA-1) has previously been affinity-purified on a column containing a G-rich telomeric DNA. LaRPA-1 binds and co-localizes with parasite telomeres in vivo. Here we describe the purification and characterization of native recombinant LaRPA-1 (rLaRPA-1). The protein was initially re-solubilized from inclusion bodies by using urea. After dialysis, rLaRPA-1 was soluble but contaminated with DNA, which was removed by an anion-exchange chromatography of the protein solubilized in urea. However, rLaRPA-1 precipitated after dialysis to remove urea. To investigate whether the contaminating DNA was involved in chaperoning the refolding of rLaRPA-1, salmon sperm DNA or heparin was added to the solution before dialysis. The addition of either of these substances prevented the precipitation of rLaRPA-1. The resulting rLaRPA-1 was soluble, correctly folded, and able to bind telomeric DNA. This is the first report showing the characterization of rLaRPA1 and of the importance of additives in chaperoning the refolding of this protein. The availability of rLaRPA-1 should be helpful in assessing the importance of this protein as a potential drug target.

  16. [Role of G-protein alpha sub-units in the morphogenic processes of filamentous Ascomycota fungi].

    PubMed

    García-Rico, Ramón O; Fierro, Francisco

    The phylum Ascomycota comprises about 75% of all the fungal species described, and includes species of medical, phytosanitary, agricultural, and biotechnological importance. The ability to spread, explore, and colonise new substrates is a feature of critical importance for this group of organisms. In this regard, basic processes such as conidial germination, the extension of hyphae and sporulation, make up the backbone of development in most filamentous fungi. These processes require specialised morphogenic machinery, coordinated and regulated by mechanisms that are still being elucidated. In recent years, substantial progress has been made in understanding the role of the signalling pathway mediated by heterotrimericG proteins in basic biological processes of many filamentous fungi. This review focuses on the role of the alpha subunits of heterotrimericG proteins in the morphogenic processes of filamentous Ascomycota.

  17. FastRNABindR: Fast and Accurate Prediction of Protein-RNA Interface Residues.

    PubMed

    El-Manzalawy, Yasser; Abbas, Mostafa; Malluhi, Qutaibah; Honavar, Vasant

    2016-01-01

    A wide range of biological processes, including regulation of gene expression, protein synthesis, and replication and assembly of many viruses are mediated by RNA-protein interactions. However, experimental determination of the structures of protein-RNA complexes is expensive and technically challenging. Hence, a number of computational tools have been developed for predicting protein-RNA interfaces. Some of the state-of-the-art protein-RNA interface predictors rely on position-specific scoring matrix (PSSM)-based encoding of the protein sequences. The computational efforts needed for generating PSSMs severely limits the practical utility of protein-RNA interface prediction servers. In this work, we experiment with two approaches, random sampling and sequence similarity reduction, for extracting a representative reference database of protein sequences from more than 50 million protein sequences in UniRef100. Our results suggest that random sampled databases produce better PSSM profiles (in terms of the number of hits used to generate the profile and the distance of the generated profile to the corresponding profile generated using the entire UniRef100 data as well as the accuracy of the machine learning classifier trained using these profiles). Based on our results, we developed FastRNABindR, an improved version of RNABindR for predicting protein-RNA interface residues using PSSM profiles generated using 1% of the UniRef100 sequences sampled uniformly at random. To the best of our knowledge, FastRNABindR is the only protein-RNA interface residue prediction online server that requires generation of PSSM profiles for query sequences and accepts hundreds of protein sequences per submission. Our approach for determining the optimal BLAST database for a protein-RNA interface residue classification task has the potential of substantially speeding up, and hence increasing the practical utility of, other amino acid sequence based predictors of protein-protein and protein

  18. A Conserved Region in the F2 Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation▿

    PubMed Central

    Gardner, Amanda E.; Dutch, Rebecca E.

    2007-01-01

    Paramyxoviruses utilize both an attachment protein and a fusion (F) protein to drive virus-cell and cell-cell fusion. F exists functionally as a trimer of two disulfide-linked subunits: F1 and F2. Alignment and analysis of a set of paramyxovirus F protein sequences identified three conserved blocks (CB): one in the fusion peptide/heptad repeat A domain, known to play important roles in fusion promotion, one in the region between the heptad repeats of F1 (CBF1) (A. E. Gardner, K. L. Martin, and R. E. Dutch, Biochemistry 46:5094-5105, 2007), and one in the F2 subunit (CBF2). To analyze the functions of CBF2, alanine substitutions at conserved positions were created in both the simian virus 5 (SV5) and Hendra virus F proteins. A number of the CBF2 mutations resulted in folding and expression defects. However, the CBF2 mutants that were properly expressed and trafficked had altered fusion promotion activity. The Hendra virus CBF2 Y79A and P89A mutants showed significantly decreased levels of fusion, whereas the SV5 CBF2 I49A mutant exhibited greatly increased cell-cell fusion relative to that for wild-type F. Additional substitutions at SV5 F I49 suggest that both side chain volume and hydrophobicity at this position are important in the folding of the metastable, prefusion state and the subsequent triggering of membrane fusion. The recently published prefusogenic structure of parainfluenza virus 5/SV5 F (H. S. Yin et al., Nature 439:38-44, 2006) places CBF2 in direct contact with heptad repeat A. Our data therefore indicate that this conserved region plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion. PMID:17507474

  19. A Method To Measure Protein Unfolding at an Air-Liquid Interface.

    PubMed

    Leiske, Danielle L; Shieh, Ian C; Tse, Martha Lovato

    2016-10-04

    Proteins are surface-active molecules that have a propensity to adsorb to hydrophobic interfaces, such as the air-liquid interface. Surface flow can increase aggregation of adsorbed proteins, which may be an undesirable consequence depending on the application. As changes in protein conformation upon adsorption are thought to induce aggregation, the ability to measure the folded state of proteins at interfaces is of particular interest. However, few techniques currently exist to measure protein conformation at interfaces. Here we describe a technique capable of measuring the hydrophobicity, and therefore the conformation and folded state, of proteins at air-liquid interfaces by exploiting the environmentally sensitive fluorophore Nile red. Two monoclonal antibodies (mAbs) with high (mAb1) and low (mAb2) surface activity were used to highlight the technique. Both mAbs showed low background fluorescence of Nile red in the liquid subphase and at a glass-liquid interface. In contrast, at the air-liquid interface Nile red fluorescence for mAb1 increased immediately after protein adsorption, whereas the Nile red fluorescence of the mAb2 film evolved more slowly in time even though the adsorbed quantity of protein remained constant. The results demonstrate that hydrophobicity upon mAb adsorption to the air-liquid interface evolves in a time-dependent manner. Interfacial hydrophobicity may be indicative of protein conformation or folded state, where rapid unfolding of mAb1 upon adsorption would be consistent with increased protein aggregation compared to mAb2. The ability to measure protein hydrophobicity at interfaces using Nile red, combined with small sample requirements and minimal sample preparation, fills a gap in existing interfacial techniques.

  20. Conservation of coevolving protein interfaces bridges prokaryote–eukaryote homologies in the twilight zone

    PubMed Central

    Rodriguez-Rivas, Juan; Marsili, Simone; Juan, David; Valencia, Alfonso

    2016-01-01

    Protein–protein interactions are fundamental for the proper functioning of the cell. As a result, protein interaction surfaces are subject to strong evolutionary constraints. Recent developments have shown that residue coevolution provides accurate predictions of heterodimeric protein interfaces from sequence information. So far these approaches have been limited to the analysis of families of prokaryotic complexes for which large multiple sequence alignments of homologous sequences can be compiled. We explore the hypothesis that coevolution points to structurally conserved contacts at protein–protein interfaces, which can be reliably projected to homologous complexes with distantly related sequences. We introduce a domain-centered protocol to study the interplay between residue coevolution and structural conservation of protein–protein interfaces. We show that sequence-based coevolutionary analysis systematically identifies residue contacts at prokaryotic interfaces that are structurally conserved at the interface of their eukaryotic counterparts. In turn, this allows the prediction of conserved contacts at eukaryotic protein–protein interfaces with high confidence using solely mutational patterns extracted from prokaryotic genomes. Even in the context of high divergence in sequence (the twilight zone), where standard homology modeling of protein complexes is unreliable, our approach provides sequence-based accurate information about specific details of protein interactions at the residue level. Selected examples of the application of prokaryotic coevolutionary analysis to the prediction of eukaryotic interfaces further illustrate the potential of this approach. PMID:27965389

  1. Structural and Functional Characterization of Cargo-Binding Sites on the μ4-Subunit of Adaptor Protein Complex 4

    PubMed Central

    Ross, Breyan H.; Lin, Yimo; Corales, Esteban A.; Burgos, Patricia V.; Mardones, Gonzalo A.

    2014-01-01

    Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (μ1-μ4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in μ subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the μ4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of μ4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type μ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered μ4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of μ4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXØ-signal of APP is limited to the non

  2. Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

    PubMed Central

    Way, Luke; Faktor, Jakub; Dvorakova, Petra; Nicholson, Judith; Vojtesek, Borek; Graham, Duncan; Ball, Kathryn L.

    2016-01-01

    Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53‐independent MDM2‐drug responsive‐binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin‐3 responsive MDM2‐binding proteins that are perturbed independent of cell density using SWATH‐MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin‐3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin‐3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin‐3 treated cells. Mitotracker confirmed that Nutlin‐3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin‐3 treatment. Proximity ligation identified rearrangements of cellular protein–protein complexes in situ. In response to Nutlin‐3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein–protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein–protein complexes in drug‐treated cells. PMID:27273042

  3. Proteins, Pathogens, and Failure at the Composite-Tooth Interface

    PubMed Central

    Spencer, P.; Ye, Q.; Misra, A.; Goncalves, S.E.P.; Laurence, J.S.

    2014-01-01

    In the United States, composites accounted for nearly 70% of the 173.2 million composite and amalgam restorations placed in 2006 (Kingman et al., 2012), and it is likely that the use of composite will continue to increase as dentists phase out dental amalgam. This trend is not, however, without consequences. The failure rate of composite restorations is double that of amalgam (Ferracane, 2013). Composite restorations accumulate more biofilm, experience more secondary decay, and require more frequent replacement. In vivo biodegradation of the adhesive bond at the composite-tooth interface is a major contributor to the cascade of events leading to restoration failure. Binding by proteins, particularly gp340, from the salivary pellicle leads to biofilm attachment, which accelerates degradation of the interfacial bond and demineralization of the tooth by recruiting the pioneer bacterium Streptococcus mutans to the surface. Bacterial production of lactic acid lowers the pH of the oral microenvironment, erodes hydroxyapatite in enamel and dentin, and promotes hydrolysis of the adhesive. Secreted esterases further hydrolyze the adhesive polymer, exposing the soft underlying collagenous dentinal matrix and allowing further infiltration by the pathogenic biofilm. Manifold approaches are being pursued to increase the longevity of composite dental restorations based on the major contributing factors responsible for degradation. The key material and biological components and the interactions involved in the destructive processes, including recent advances in understanding the structural and molecular basis of biofilm recruitment, are described in this review. Innovative strategies to mitigate these pathogenic effects and slow deterioration are discussed. PMID:25190266

  4. Neutralizing Monoclonal Antibodies against Ricin’s Enzymatic Subunit Interfere with Protein Disulfide Isomerase-Mediated Reduction of Ricin Holotoxin In Vitro

    PubMed Central

    O’Hara, Joanne M.; Mantis, Nicholas J.

    2013-01-01

    The penultimate event in the intoxication of mammalian cells by ricin toxin is the reduction, in the endoplasmic reticulum (ER), of the intermolecular disulfide bond that links ricin’s enzymatic (RTA) and binding (RTB) subunits. In this report we adapted an in vitro protein disulfide isomerase (PDI)-mediated reduction assay to test the hypothesis that the RTA-specific neutralizing monoclonal antibody (mAb) IB2 interferes with the liberation of RTA from RTB. IB2 recognizes an epitope located near the interface between RTA and RTB and, like a number of other RTA-specific neutralizing mAbs, is proposed to neutralize ricin intracellularly. In this study, we found that IB2 virtually eliminated the reduction of ricin holotoxin into RTA and RTB in vitro. Surprisingly, three other neutralizing mAbs (GD12, R70 and SyH7) that bind epitopes at considerable distance from ricin’s disulfide bond were as effective (or nearly as effective) as IB2 in interfering with PDI-mediated liberation of RTA from RTB. By contrast, two non-neutralizing RTA-specific mAbs, FGA12 and SB1, did not affect PDI-mediated reduction of ricin. These data reveal a possible mechanism by which RTA-specific antibodies may neutralize ricin intracellularly, provided they are capable of trafficking in association with ricin from the cell surface to the ER. PMID:23774033

  5. Cellular protein and mRNA expression of β1 nicotinic acetylcholine receptor (nAChR) subunit in brain, skeletal muscle and placenta.

    PubMed

    Aishah, Atqiya; Hinton, Tina; Machaalani, Rita

    2017-01-30

    The β1 nicotinic acetylcholine receptor (nAChR) subunit is a muscle type subunit of this family and as such, is found predominantly in muscle. Recent reports document its expression in other tissues and cell lines including adrenal glands, carcinomas, lung and brain. However, the majority of studies were of tissue lysates, thus the cellular distribution was not determined. This study aimed to determine the cellular distribution of the β1 nAChR subunit in the brain, at both the mRNA and protein levels, using non-radioactive in situ hybridization (ISH) and immunohistochemistry (IHC), respectively, and to compare it to two muscle tissue types, skeletal and placenta. Tissue was formalin fixed and paraffin embedded (all tissue types) and frozen (placenta) from humans. Additional control tissue from the piglet and mouse brain were also studied, as was mRNA for the α3 nAChR and N-methyl-d-aspartate receptor 1 (NR1) subunit. We found no β1 nAChR subunit mRNA expression in the human and piglet brain despite strong protein expression. Some signal was seen in the mouse brain but considered inconclusive given the probes designed were not of 100% homology to the mouse. In the skeletal muscle and placenta tissues, β1 nAChR subunit mRNA expression was prominent and mirrored protein expression. No α3 nAChR or NR1 mRNA was seen in the skeletal muscle, as expected, although both subunit mRNAs were present in the placenta. This study concludes that further experiments are required to conclusively state that the β1 nAChR subunit is expressed in the human, piglet and mouse brain.

  6. Control of the ATP synthase beta subunit expression by RNA-binding proteins TIA-1, TIAR, and HuR.

    PubMed

    Izquierdo, José M

    2006-09-22

    The beta-subunit of the mitochondrial H+-ATP synthase (beta-F1-ATPase) catalyzes the rate-limiting step of ATP formation in eukaryotic cells. Here, we examined the post-transcriptional regulation of human beta-F1-ATPase mediated by the 3'-untranslated region of the mRNA (beta-3'-UTR). Biochemical analysis revealed that the adenosine/uridine (AU)-rich element-binding proteins TIA-1 (T-cell intracellular antigen-1), TIAR (TIA-1-related protein), and HuR (Hu antigen R) interact with the beta-F1-ATPase mRNA through an AU-rich sequence located to the 3'-UTR. Mouse embryonic fibroblasts (MEFs) knocked-out for TIA-1 or RNA interference (RNAi)-mediated knockdown of endogenous TIA-1, TIAR, or HuR in HeLa cells resulted in a decrease in beta-F1-ATPase protein expression. The expression of GFP from a chimeric reporter containing human beta-3'-UTR was also abolished in HeLa cells depleted of TIA-1, TIAR, or HuR. MEFs knocked-in for TIA-1 or the overexpression of RNAi-resistant TIA-1, TIAR, or HuR proteins in the RNAi-treated HeLa cells significantly restored the levels of the expression of both endogenous mouse beta-F1-ATPase protein or recombinant GFP.

  7. Complex stability and dynamic subunit interchange modulates the disparate activities of the yeast moonlighting proteins Hal3 and Vhs3

    PubMed Central

    Abrie, J. Albert; Molero, Cristina; Ariño, Joaquín; Strauss, Erick

    2015-01-01

    Saccharomyces cerevisiae Hal3 and Vhs3 are moonlighting proteins, acting both as inhibitors of the serine/threonine protein phosphatase Ppz1 and as subunits (together with Cab3) of the unique heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme of Hemiascomycetous yeast. Both these roles are essential: PPCDC catalyses the third step of coenzyme A biosynthesis, while Ppz1 inhibition is required for regulation of monovalent cation homeostasis. However, the mechanisms by which these proteins’ disparate activities are regulated are not well understood. The PPCDC domains (PDs) of Hal3, Vhs3 and Cab3 constitute the minimum requirement for these proteins to show both PPCDC activity and, in the case of Hal3 and Vhs3, to bind to Ppz1. Using these PD proteins as a model system to study the possibility of dynamic interchange between these roles, we provide evidence that Hal3 binds Ppz1 as a monomer (1:1 stoichiometry), requiring it to de-oligomerize from its usual homo- and heterotrimeric states (the latter having PPCDC activity). This de-oligomerization is made possible by structural features that set Hal3 apart from Vhs3, increasing its ability to undergo monomer exchange. These findings suggest that oligomer interchange may be a significant factor in the functional regulation of these proteins and their various unrelated (moonlighting) functions. PMID:26514574

  8. Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

    SciTech Connect

    Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.; E-mail: andy.blakely@vanderbilt.edu

    2005-08-05

    The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH{sub 2}-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking.

  9. Characterization of interfacial solvent in protein complexes and contribution of wet spots to the interface description.

    PubMed

    Teyra, Joan; Pisabarro, M T

    2007-06-01

    Water networks in protein interfaces can complement direct interactions contributing significantly to molecular recognition, function, and stability of protein association. Thus, water can be seen as an extension or addition of protein structural features, which may add plenty of information to protein interfacial definition. However, solvent is frequently neglected in protein interaction studies. Analysis of the interfacial information contained in the PDB is essential to achieve more accurate descriptions of protein interfaces. With this aim, we have used the SCOWLP database (http://www.scowlp.org) and applied computational geometry methods to extract and analyze interfacial information of a high-resolution nonredundant dataset of 176 protein complexes containing obligate and transient interfaces. We have identified all interfacial residues and characterized them in terms of temperature factors, secondary structure, residue composition, and pairing preferences to understand their contribution to the interface description. We have paid special attention to water-bridged residues; focusing on those that interact only mediated by a water molecule called wet spots. Our results show that 40.1% of the interfacial residues are interacting through water and that wet spots represent a 14.5% of the total, emphasizing the importance of the inclusion of solvent in protein interaction studies, and the contribution of wet spots to interfacial description. Wet spots present similar characteristics to residues binding buried water molecules in the core or cavities of proteins; being preferably located in nonregular secondary structures and establishing hydrogen bonds by their main-chains. We observe that obligate and transient interfaces present a comparable amount of solvent. Moreover, the role of solvent in both complex types differs according to the different nature of their interfaces. The information obtained in our studies will assist in the process of accomplishing more

  10. Binomial distribution for quantification of protein subunits in biological nanoassemblies and functional nanomachines.

    PubMed

    Fang, Huaming; Zhang, Peng; Huang, Lisa P; Zhao, Zhengyi; Pi, Fengmei; Montemagno, Carlo; Guo, Peixuan

    2014-10-01

    Living systems produce ordered structures and nanomachines that inspire the development of biomimetic nanodevices such as chips, MEMS, actuators, sensors, sorters, and apparatuses for single-pore DNA sequencing, disease diagnosis, drug or therapeutic RNA delivery. Determination of the copy numbers of subunits that build these machines is challenging due to small size. Here we report a simple mathematical method to determine the stoichiometry, using phi29 DNA-packaging nanomotor as a model to elucidate the application of a formula ∑M=0(Z)((Z)M)p(Z-M)q(M), where p and q are the percentage of wild-type and inactive mutant in the empirical assay; M is the copy numbers of mutant and Z is the stoichiometry in question. Variable ratios of mutants and wild-type were mixed to inhibit motor function. Empirical data were plotted over the theoretical curves to determine the stoichiometry and the value of K, which is the number of mutant needed in each machine to block the function, all based on the condition that wild-type and mutant are equal in binding affinity. Both Z and K from 1-12 were investigated. The data precisely confirmed that phi29 motor contains six copies (Z) of the motor ATPase gp16, and K=1. From the clinical editor: To determine copy numbers of subunits that form nanomachines in living organisms is a daunting task due to the complexities and the inherently small sizes associated with such systems. In this paper, a simple mathematical method is described how to determine the stoichiometry of copies in biomimetic nanodevices, using phi29 DNA-packaging nanomotor as a model.

  11. β-subunit myristoylation functions as an energy sensor by modulating the dynamics of AMP-activated Protein Kinase

    PubMed Central

    Ali, Nada; Ling, Naomi; Krishnamurthy, Srinath; Oakhill, Jonathan S.; Scott, John W.; Stapleton, David I.; Kemp, Bruce E.; Anand, Ganesh Srinivasan; Gooley, Paul R.

    2016-01-01

    The heterotrimeric AMP-activated protein kinase (AMPK), consisting of α, β and γ subunits, is a stress-sensing enzyme that is activated by phosphorylation of its activation loop in response to increases in cellular AMP. N-terminal myristoylation of the β-subunit has been shown to suppress Thr172 phosphorylation, keeping AMPK in an inactive state. Here we use amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the structural and dynamic properties of the mammalian myristoylated and non-myristoylated inactivated AMPK (D139A) in the presence and absence of nucleotides. HDX MS data suggests that the myristoyl group binds near the first helix of the C-terminal lobe of the kinase domain similar to other kinases. Our data, however, also shows that ATP.Mg2+ results in a global stabilization of myristoylated, but not non-myristoylated AMPK, and most notably for peptides of the activation loop of the α-kinase domain, the autoinhibitory sequence (AIS) and the βCBM. AMP does not have that effect and HDX measurements for myristoylated and non-myristoylated AMPK in the presence of AMP are similar. These differences in dynamics may account for a reduced basal rate of phosphorylation of Thr172 in myristoylated AMPK in skeletal muscle where endogenous ATP concentrations are very high. PMID:28000716

  12. Development of a subunit vaccine containing recombinant Riemerella anatipestifer outer membrane protein A and CpG ODN adjuvant.

    PubMed

    Chu, Chun-Yen; Liu, Chia-Hui; Liou, Jhong-Jie; Lee, Jai-Wei; Cheng, Li-Ting

    2015-01-01

    Riemerella anatipestifer, a Gram-negative bacillus, causes septicemia that can result in high mortality for ducklings. In this study, we evaluated the immune response and protective efficacy provided by a subunit vaccine containing recombinant outer membrane protein A (rOmpA) and plasmid constructs containing CpG oligodeoxynucleotides (ODN). Results showed that CpG ODN enhanced both humoral and cell-mediated immunity elicited by rOmpA as early as two weeks after primary immunization. When compared to ducks immunized with rOmpA, ducks immunized with rOmpA+CpG ODN showed higher levels (p<0.05) of antibody titer, T cell proliferation, and percentages of CD4(+) and CD8(+) T cell in peripheral blood mononuclear cells (PBMCs). The relative fold inductions of mRNA expression of Th1-type (IFN-γ and IL-12), and Th2-type (IL-6) cytokines in PBMCs isolated from ducks immunized with rOmpA+CpG ODN were significantly higher than those of the rOmpA group. Homologous challenge result showed that the rOmpA+CpG ODN vaccine reduced the pathological score by 90% in comparison with the saline control. In conclusion, our study found that CpG ODN can enhance both humoral and cellular immunity elicited by a rOmpA vaccine. The rOmpA+CpG ODN vaccine can be further developed as a subunit vaccine against R. anatipestifer.

  13. Differential dependence of the D1 and D5 dopamine receptors on the G protein gamma 7 subunit for activation of adenylylcyclase.

    PubMed

    Wang, Q; Jolly, J P; Surmeier, J D; Mullah, B M; Lidow, M S; Bergson, C M; Robishaw, J D

    2001-10-19

    The D(1) dopamine receptor, G protein gamma(7) subunit, and adenylylcyclase are selectively expressed in the striatum, suggesting their potential interaction in a common signaling pathway. To evaluate this possibility, a ribozyme strategy was used to suppress the expression of the G protein gamma(7) subunit in HEK 293 cells stably expressing the human D(1) dopamine receptor. Prior in vitro analysis revealed that the gamma(7) ribozyme possessed cleavage activity directed exclusively toward the gamma(7) RNA transcript (Wang, Q., Mullah, B., Hansen, C., Asundi, J., and Robishaw, J. D. (1997) J. Biol. Chem. 272, 26040-26048). In vivo analysis of cells transfected with the gamma(7) ribozyme showed a specific reduction in the expression of the gamma(7) protein. Coincident with the loss of the gamma(7) protein, there was a noticeable reduction in the expression of the beta(1) protein, confirming their interaction in these cells. Finally, functional analysis of ribozyme-mediated suppression of the beta(1) and gamma(7) proteins revealed a significant attenuation of SKF81297-stimulated adenylylcyclase activity in D(1) dopamine receptor-expressing cells. By contrast, ribozyme-mediated suppression of the beta(1) and gamma(7) proteins showed no reduction of SKF81297-stimulated adenylylcyclase activity in D(5) dopamine receptor-expressing cells. Taken together, these data indicate that the structurally related D(1) and D(5) dopamine receptor subtypes utilize G proteins composed of distinct betagamma subunits to stimulate adenylylcyclase in HEK 293 cells. Underscoring the physiological relevance of these findings, single cell reverse transcriptase-polymerase chain reaction analysis revealed that the D(1) dopamine receptor and the G protein gamma(7) subunit are coordinately expressed in substance P containing neurons in rat striatum, suggesting that the G protein gamma(7) subunit may be a new target for drugs to selectively alter dopaminergic signaling within the brain.

  14. Ascertaining effects of nanoscale polymeric interfaces on competitive protein adsorption at the individual protein level

    NASA Astrophysics Data System (ADS)

    Song, Sheng; Xie, Tian; Ravensbergen, Kristina; Hahm, Jong-In

    2016-02-01

    With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those occurring in a competitive adsorption environment. Complex sequences of adhesion events in competitive adsorption involving multicomponent protein systems have been extensively investigated, but our understanding is still limited primarily to macroscopic adhesion onto chemically simple surfaces. We examine the competitive adsorption behavior from a binary protein mixture containing bovine serum albumin and fibrinogen at the single protein level. We subsequently evaluate a series of adsorption and displacement processes occurring on both the macroscopic homopolymer and nanoscopic diblock copolymer surfaces, while systematically varying the protein concentration and incubation time. We identify the similarities and dissimilarities in competitive protein adsorption behavior between the two polymeric surfaces, the former presenting chemical uniformity at macroscale versus the latter exhibiting periodic nanointerfaces of chemically alternating polymeric segments. We then present our novel experimental finding of a large increase in the nanointerface-engaged residence time of the initially bound proteins and further explain the origin of this phenomenon manifested on nanoscale diblock copolymer surfaces. The outcomes of this study may provide timely insight into nanoscale competitive protein adsorption that is much needed in designing bioimplant and tissue engineering materials. In addition, the fundamental understanding gained from this study can be beneficial for the development of highly miniaturized biodevices and biomaterials fabricated by using nanoscale polymeric materials and interfaces.With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those

  15. Structural sequences are conserved in the genes coding for the alpha, alpha' and beta-subunits of the soybean 7S seed storage protein.

    PubMed Central

    Schuler, M A; Ladin, B F; Pollaco, J C; Freyer, G; Beachy, R N

    1982-01-01

    Cloned DNAs encoding four different proteins have been isolated from recombinant cDNA libraries constructed with Glycine max seed mRNAs. Two cloned DNAs code for the alpha and alpha'-subunits of the 7S seed storage protein (conglycinin). The other cloned cDNAs code for proteins which are synthesized in vitro as 68,000 d., 60,000 d. or 53,000 d. polypeptides. Hybrid selection experiments indicate that, under low stringency hybridization conditions, all four cDNAs hybridize with mRNAs for the alpha and alpha'-subunits and the 68,000 d., 60,000 d. and 53,000 d. in vitro translation products. Within three of the mRNA, there is a conserved sequence of 155 nucleotides which is responsible for this hybridization. The conserved nucleotides in the alpha and alpha'-subunit cDNAs and the 68,000 d. polypeptide cDNAs span both coding and noncoding sequences. The differences in the coding nucleotides outside the conserved region are extensive. This suggests that selective pressure to maintain the 155 conserved nucleotides has been influenced by the structure of the seed mRNA. RNA blot hybridizations demonstrate that mRNA encoding the other major subunit (beta) of the 7S seed storage protein also shares sequence homology with the conserved 155 nucleotide sequence of the alpha and alpha'-subunit mRNAs, but not with other coding sequences. Images PMID:6897678

  16. Understanding the fabric of protein crystals: computational classification of biological interfaces and crystal contacts

    PubMed Central

    Capitani, Guido; Duarte, Jose M.; Baskaran, Kumaran; Bliven, Spencer; Somody, Joseph C.

    2016-01-01

    Modern structural biology still draws the vast majority of information from crystallography, a technique where the objects being investigated are embedded in a crystal lattice. Given the complexity and variety of those objects, it becomes fundamental to computationally assess which of the interfaces in the lattice are biologically relevant and which are simply crystal contacts. Since the mid-1990s, several approaches have been applied to obtain high-accuracy classification of crystal contacts and biological protein–protein interfaces. This review provides an overview of the concepts and main approaches to protein interface classification: thermodynamic estimation of interface stability, evolutionary approaches based on conservation of interface residues, and co-occurrence of the interface across different crystal forms. Among the three categories, evolutionary approaches offer the strongest promise for improvement, thanks to the incessant growth in sequence knowledge. Importantly, protein interface classification algorithms can also be used on multimeric structures obtained using other high-resolution techniques or for protein assembly design or validation purposes. A key issue linked to protein interface classification is the identification of the biological assembly of a crystal structure and the analysis of its symmetry. Here, we highlight the most important concepts and problems to be overcome in assembly prediction. Over the next few years, tools and concepts of interface classification will probably become more frequently used and integrated in several areas of structural biology and structural bioinformatics. Among the main challenges for the future are better addressing of weak interfaces and the application of interface classification concepts to prediction problems like protein–protein docking. Supplementary information: Supplementary data are available at Bioinformatics online. Contact: guido.capitani@psi.ch PMID:26508758

  17. Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

    SciTech Connect

    Tai, Lin-Ru; Chou, Chang-Wei; Wu, Jing-Ying; Kirby, Ralph; Lin, Alan

    2013-11-15

    Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20{sub NLS} mutant gene and examined polysome profile of cells that had been transfected with the S20{sub NLS} gene. As a result, we observed the formation of recombinant 40S carried S20{sub NLS} but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20{sub NLS} in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20{sub NLS} in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20{sub NLS} is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20{sub NLS}. • Cytoplasm-retained S20{sub NLS} is crucial for creating a functional small subunit.

  18. Ion-specific induced fluctuations and free energetics of aqueous protein hydrophobic interfaces: toward connecting to specific-ion behaviors at aqueous liquid-vapor interfaces.

    PubMed

    Cui, Di; Ou, Shuching; Peters, Eric; Patel, Sandeep

    2014-05-01

    We explore anion-induced interface fluctuations near protein-water interfaces using coarse-grained representations of interfaces as proposed by Willard and Chandler ( J. Phys. Chem. B 2010 , 114 , 1954 - 1958 ). We use umbrella sampling molecular dynamics to compute potentials of mean force along a reaction coordinate bridging the state where the anion is fully solvated and one where it is biased via harmonic restraints to remain at the protein-water interface. Specifically, we focus on fluctuations of an interface between water and a hydrophobic region of hydrophobin-II (HFBII), a 71 amino acid residue protein expressed by filamentous fungi and known for its ability to form hydrophobically mediated self-assemblies at interfaces such as a water/air interface. We consider the anions chloride and iodide that have been shown previously by simulations as displaying specific-ion behaviors at aqueous liquid-vapor interfaces. We find that as in the case of a pure liquid-vapor interface, at the hydrophobic protein-water interface, the larger, less charge-dense iodide anion displays a marginal interfacial stability compared with that of the smaller, more charge-dense chloride anion. Furthermore, consistent with the results at aqueous liquid-vapor interfaces, we find that iodide induces larger fluctuations of the protein-water interface than chloride.

  19. Interactions between the human RNA polymerase II subunits.

    PubMed

    Acker, J; de Graaff, M; Cheynel, I; Khazak, V; Kedinger, C; Vigneron, M

    1997-07-04

    As an initial approach to characterizing the molecular structure of the human RNA polymerase II (hRPB), we systematically investigated the protein-protein contacts that the subunits of this enzyme may establish with each other. To this end, we applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect cells, which were coinfected with all possible combinations of recombinant baculoviruses expressing hRPB subunits, either as untagged polypeptides or as glutathione S-transferase fusion proteins. This is the first comprehensive study of interactions between eukaryotic RNA polymerase subunits; among the 116 combinations of hRPB subunits tested, 56 showed significant to strong interactions, whereas 60 were negative. Within the intricate network of interactions, subunits hRPB3 and hRPB5 play a central role in polymerase organization. These subunits, which are able to homodimerize and to interact, may constitute the nucleation center for polymerase assembly, by providing a large interface to most of the other subunits.

  20. Pyruvate dehydrogenase complex: mRNA and protein expression patterns of E1α subunit genes in human spermatogenesis.

    PubMed

    Pinheiro, Ana; Silva, Maria João; Graça, Inês; Silva, Joaquina; Sá, Rosália; Sousa, Mário; Barros, Alberto; Tavares de Almeida, Isabel; Rivera, Isabel

    2012-09-10

    During spermatogenesis, germ cells undergo a complex process of cell differentiation and morphological restructuring, which depends on the coordinated expression of different genes. Some vital examples are those involved in cell energy metabolism, namely the genes encoding the E1α subunit of pyruvate dehydrogenase complex: the somatic PDHA1 (X-linked) and the testis-specific PDHA2 (autosomal). There are no data related to the study at the RNA and protein levels of PDHA genes during human spermatogenesis. The present study aimed to describe the mRNA and protein expression patterns of the human PDHA genes during spermatogenesis. Expression profiles of the PDHA1 and PDHA2 genes were characterized using different human tissues and cells. Diploid and haploid germ cells fractions were obtained from testis tissues. The mRNA profiles were analyzed by quantitative RT-PCR, whereas the protein profiles were evaluated by immunohistochemistry, western blotting and two-dimensional electrophoresis. Expression of the PDHA1 gene was found in all somatic cells, whereas expression of PDHA2 gene was restricted to germ cells. The switch from X-linked to autosomic gene expression occurred in spermatocytes. Data suggest the activation of PDHA2 gene expression is most probably a mechanism to ensure the continued expression of the protein, thus allowing germ cell viability and functionality.

  1. Secondary Structure and Subunit Composition of Soy Protein In Vitro Digested by Pepsin and Its Relation with Digestibility

    PubMed Central

    Yang, Yong; Wang, Zhongjiang; Wang, Rui; Sui, Xiaonan; Qi, Baokun; Han, Feifei; Li, Yang; Jiang, Lianzhou

    2016-01-01

    In the present study, in vitro digestibility and structure of soybean protein isolates (SPIs) prepared from five soybean varieties were investigated in simulated gastric fluid (SGF), using FT-IR microspectroscopy and SDS-PAGE. The result indicated that β-conformations were prone to be hydrolyzed by pepsin preferentially and transformed to unordered structure during in vitro digestion, followed by the digestion of α-helix and unordered structure. A negative linear correlation coefficient was found between the β-conformation contents of five SPIs and their in vitro digestibility values. The intensities of the protein bands corresponding to 7S and 11S fractions were decreased and many peptide bands appeared at 11~15 kDa during enzymatic hydrolysis. β-conglycinin was poorly hydrolyzed with pepsin, especially the β-7S subunit. On the other hand, basic polypeptides of glycinin degraded slower than acidic polypeptides and represented a large proportion of the residual protein after digestion. 11S-A3 of all SPIs disappeared after 1 h digestion. Moreover, a significant negative linear correlation coefficient (r = −0.89) was found between the β-7S contents of five SPIs and their in vitro digestibility values. These results are useful for further studies of the functional properties and bioactive properties of these varieties and laid theoretical foundations for the development of the specific functional soy protein isolate. PMID:27298825

  2. Mapping the domain structure of the influenza A virus polymerase acidic protein (PA) and its interaction with the basic protein 1 (PB1) subunit

    SciTech Connect

    Guu, Tom S.Y.; Dong Liping; Wittung-Stafshede, Pernilla; Tao, Yizhi J.

    2008-09-15

    The influenza A virus polymerase consists of three subunits (PA, PB1, and PB2) necessary for viral RNA synthesis. The heterotrimeric polymerase complex forms through PA interacting with PB1 and PB1 interacting with PB2. PA has been shown to play critical roles in the assembly, catalysis, and nuclear localization of the polymerase. To probe the structure of PA, we isolated recombinant PA from insect cells. Limited proteolysis revealed that PA contained two domains connected by a 20-residue linker (residues 257-276). Far-UV circular dichroism established that the two domains folded into a mixed {alpha}/{beta} structure when separately expressed. In vitro pull-down assays showed that neither individually nor cooperatively expressed PA domains, without the linker, could assure PA-PB1 interaction. Protease treatment of PA-PB1 complex indicated that its PA subunit was significantly more stable than free PA, suggesting that the linker is protected and it constitutes an essential component of the PA-PB1 interface.

  3. Intermolecular Interaction between Anchoring Subunits Specify Subcellular Targeting and Function of RGS Proteins in Retina ON-Bipolar Neurons.

    PubMed

    Sarria, Ignacio; Orlandi, Cesare; McCall, Maureen A; Gregg, Ronald G; Martemyanov, Kirill A

    2016-03-09

    In vertebrate retina, light responses generated by the rod photoreceptors are transmitted to the second-order neurons, the ON-bipolar cells (ON-BC), and this communication is indispensible for vision in dim light. In ON-BCs, synaptic transmission is initiated by the metabotropic glutamate receptor, mGluR6, that signals via the G-protein Go to control opening of the effector ion channel, TRPM1. A key role in this process belongs to the GTPase Activating Protein (GAP) complex that catalyzes Go inactivation upon light-induced suppression of glutamate release in rod photoreceptors, thereby driving ON-BC depolarization to changes in synaptic input. The GAP complex has a striking molecular complexity. It contains two Regulator of G-protein Signaling (RGS) proteins RGS7 and RGS11 that directly act on Go and two adaptor subunits: RGS Anchor Protein (R9AP) and the orphan receptor, GPR179. Here we examined the organizational principles of the GAP complex in ON-BCs. Biochemical experiments revealed that RGS7 binds to a conserved site in GPR179 and that RGS11 in vivo forms a complex only with R9AP. R9AP and GPR179 are further integrated via direct protein-protein interactions involving their cytoplasmic domains. Elimination of GPR179 prevents postsynaptic accumulation of R9AP. Furthermore, concurrent knock-out of both R9AP and RGS7 does not reconfigure the GAP complex and completely abolishes synaptic transmission, resulting in a novel mouse model of night blindness. Based on these results, we propose a model of hierarchical assembly and function of the GAP complex that supports ON-BCs visual signaling.

  4. Radiolabeled Adenoviral Sub-unit Proteins for Molecular Imaging and Therapeutic Applications in Oncology

    SciTech Connect

    Srivastava, S.; Meinken, G.; Springer, K. Awasthi, V.; Freimuth, P.

    2004-10-06

    The objective of this project was to develop and optimize new ligand systems, based on adenoviral vectors (intact adenovirus, adeno-viral fiber protein, and the knob protein), for delivering suitable radionuclides into tumor cells for molecular imaging and combined gene/radionuclide therapy of cancer.

  5. Destabilization of the MutSα’s protein-protein interface due to binding to the DNA adduct induced by anticancer agent Carboplatin via molecular dynamics simulations

    PubMed Central

    Negureanu, Lacramioara; Salsbury, Freddie R

    2013-01-01

    DNA mismatch repair (MMR) proteins maintain genetic integrity in all organisms by recognizing and repairing DNA errors. Such alteration of hereditary information can lead to various diseases, including cancer. Besides their role in DNA repair, MMR proteins detect and initiate cellular responses to certain type of DNA damage. Its response to the damaged DNA has made the human MMR pathway a useful target for anticancer agents such as carboplatin. This study indicates that strong, specific interactions at the interface of MutSα in response to the mismatched DNA recognition are replaced by weak, non-specific interactions in response to the damaged DNA recognition. Data suggest a severe impairment of the dimerization of MutSα in response to the damaged DNA recognition. While the core of MutSα is preserved in response to the damaged DNA recognition, the loss of contact surface and the rearrangement of contacts at the protein interface suggest a different packing in response to the damaged DNA recognition. Coupled in response to the mismatched DNA recognition, interaction energies, hydrogen bonds, salt bridges, and solvent accessible surface areas at the interface of MutSα and within the subunits are uncoupled or asynchronously coupled in response to the damaged DNA recognition. These pieces of evidence suggest that the loss of a synchronous mode of response in the MutSα’s surveillance for DNA errors would possible be one of the mechanism(s) of signaling the MMR-dependent programed cell death much wanted in anticancer therapies. The analysis was drawn from dynamics simulations. PMID:24061854

  6. A Pan-Cancer Catalogue of Cancer Driver Protein Interaction Interfaces.

    PubMed

    Porta-Pardo, Eduard; Garcia-Alonso, Luz; Hrabe, Thomas; Dopazo, Joaquin; Godzik, Adam

    2015-10-01

    Despite their importance in maintaining the integrity of all cellular pathways, the role of mutations on protein-protein interaction (PPI) interfaces as cancer drivers has not been systematically studied. Here we analyzed the mutation patterns of the PPI interfaces from 10,028 proteins in a pan-cancer cohort of 5,989 tumors from 23 projects of The Cancer Genome Atlas (TCGA) to find interfaces enriched in somatic missense mutations. To that end we use e-Driver, an algorithm to analyze the mutation distribution of specific protein functional regions. We identified 103 PPI interfaces enriched in somatic cancer mutations. 32 of these interfaces are found in proteins coded by known cancer driver genes. The remaining 71 interfaces are found in proteins that have not been previously identified as cancer drivers even that, in most cases, there is an extensive literature suggesting they play an important role in cancer. Finally, we integrate these findings with clinical information to show how tumors apparently driven by the same gene have different behaviors, including patient outcomes, depending on which specific interfaces are mutated.

  7. Adsorption of the natural protein surfactant Rsn-2 onto liquid interfaces.

    PubMed

    Brandani, Giovanni B; Vance, Steven J; Schor, Marieke; Cooper, Alan; Kennedy, Malcolm W; Smith, Brian O; MacPhee, Cait E; Cheung, David L

    2017-03-22

    To stabilize foams, droplets and films at liquid interfaces a range of protein biosurfactants have evolved in nature. Compared to synthetic surfactants, these combine surface activity with biocompatibility and low solution aggregation. One recently studied example is Rsn-2, a component of the foam nest of the frog Engystomops pustulosus, which has been predicted to undergo a clamshell-like opening transition at the air-water interface. Using atomistic molecular dynamics simulations and surface tension measurements we study the adsorption of Rsn-2 onto air-water and cyclohexane-water interfaces. The protein adsorbs readily at both interfaces, with adsorption mediated by the hydrophobic N-terminus. At the cyclohexane-water interface the clamshell opens, due to the favourable interaction between hydrophobic residues and cyclohexane molecules and the penetration of cyclohexane molecules into the protein core. Simulations of deletion mutants showed that removal of the N-terminus inhibits interfacial adsorption, which is consistent with the surface tension measurements. Deletion of the hydrophilic C-terminus also affects adsorption, suggesting that this plays a role in orienting the protein at the interface. The characterisation of the interfacial behaviour gives insight into the factors that control the interfacial adsorption of proteins, which may inform new applications of this and similar proteins in areas including drug delivery and food technology and may also be used in the design of synthetic molecules showing similar changes in conformation at interfaces.

  8. A Pan-Cancer Catalogue of Cancer Driver Protein Interaction Interfaces

    PubMed Central

    Hrabe, Thomas; Dopazo, Joaquin; Godzik, Adam

    2015-01-01

    Despite their importance in maintaining the integrity of all cellular pathways, the role of mutations on protein-protein interaction (PPI) interfaces as cancer drivers has not been systematically studied. Here we analyzed the mutation patterns of the PPI interfaces from 10,028 proteins in a pan-cancer cohort of 5,989 tumors from 23 projects of The Cancer Genome Atlas (TCGA) to find interfaces enriched in somatic missense mutations. To that end we use e-Driver, an algorithm to analyze the mutation distribution of specific protein functional regions. We identified 103 PPI interfaces enriched in somatic cancer mutations. 32 of these interfaces are found in proteins coded by known cancer driver genes. The remaining 71 interfaces are found in proteins that have not been previously identified as cancer drivers even that, in most cases, there is an extensive literature suggesting they play an important role in cancer. Finally, we integrate these findings with clinical information to show how tumors apparently driven by the same gene have different behaviors, including patient outcomes, depending on which specific interfaces are mutated. PMID:26485003

  9. Broad-Bandwidth Chiral Sum Frequency Generation Spectroscopy for Probing the Kinetics of Proteins at Interfaces

    PubMed Central

    2016-01-01

    The kinetics of proteins at interfaces plays an important role in biological functions and inspires solutions to fundamental problems in biomedical sciences and engineering. Nonetheless, due to the lack of surface-specific and structural-sensitive biophysical techniques, it still remains challenging to probe protein kinetics in situ and in real time without the use of spectroscopic labels at interfaces. Broad-bandwidth chiral sum frequency generation (SFG) spectroscopy has been recently developed for protein kinetic studies at interfaces by tracking the chiral vibrational signals of proteins. In this article, we review our recent progress in kinetic studies of proteins at interfaces using broad-bandwidth chiral SFG spectroscopy. We illustrate the use of chiral SFG signals of protein side chains in the C–H stretch region to monitor self-assembly processes of proteins at interfaces. We also present the use of chiral SFG signals from the protein backbone in the N–H stretch region to probe the real-time kinetics of proton exchange between protein and water at interfaces. In addition, we demonstrate the applications of spectral features of chiral SFG that are typical of protein secondary structures in both the amide I and the N–H stretch regions for monitoring the kinetics of aggregation of amyloid proteins at membrane surfaces. These studies exhibit the power of broad-bandwidth chiral SFG to study protein kinetics at interfaces and the promise of this technique in research areas of surface science to address fundamental problems in biomedical and material sciences. PMID:26196215

  10. Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

    PubMed Central

    Chung, T D; Wymer, J P; Smith, C C; Kulka, M; Aurelian, L

    1989-01-01

    The large subunit of the herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (RR1) is demonstrated to possess serine/threonine-specific kinase activity. Computer-assisted sequence analysis identified regions within the amino terminus of ICP10 that are homologous to the catalytic domain of known protein kinases (PKs). An in vitro kinase assay confirmed the ability of ICP10, immunoprecipitated from either HSV-2-infected cells or from cells transfected with an ICP10 expression vector, to autophosphorylate and transphosphorylate exogenous substrates in the presence of ATP and Mg2+. The HSV-1 RR1 was shown to be negative for PK activity under these conditions. PK activity was localized to a 57-kDa amino-terminal region within ICP10 that lacked RR activity. Images PMID:2545912

  11. Metallothionein 2A affects the cell respiration by suppressing the expression of mitochondrial protein cytochrome c oxidase subunit II.

    PubMed

    Bragina, Olga; Gurjanova, Karina; Krishtal, Jekaterina; Kulp, Maria; Karro, Niina; Tõugu, Vello; Palumaa, Peep

    2015-06-01

    Metallothioneins (MT) are involved in a broad range of cellular processes and play a major role in protection of cells towards various stressors. Two functions of MTs, namely the maintaining of the homeostasis of transition metal ions and the redox balance, are directly linked to the functioning of mitochondria. Dyshomeostasis of MTs is often related with malfunctioning of mitochondria; however, the mechanism by which MTs affect the mitochondrial respiratory chain is still unknown. We demonstrated that overexpression of MT-2A in HEK cell line decreased the oxidative phosphorylation capacity of the cells. HEK cells overexpressing MT-2A demonstrated reduced oxygen consumption and lower cellular ATP levels. MT-2A did not affect the number of mitochondria, but reduced specifically the level of cytochrome c oxidase subunit II protein, which resulted in lower activity of the complex IV.

  12. Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding.

    PubMed

    Herberg, F W; Maleszka, A; Eide, T; Vossebein, L; Tasken, K

    2000-04-28

    Compartmentalization of cAMP-dependent protein kinase (PKA) is in part mediated by specialized protein motifs in the dimerization domain of the regulatory (R)-subunits of PKA that participate in protein-protein interactions with an amphipathic helix region in A-kinase anchoring proteins (AKAPs). In order to develop a molecular understanding of the subcellular distribution and specific functions of PKA isozymes mediated by association with AKAPs, it is of importance to determine the apparent binding constants of the R-subunit-AKAP interactions. Here, we present a novel approach using surface plasmon resonance (SPR) to examine directly the association and dissociation of AKAPs with all four R-subunit isoforms immobilized on a modified cAMP surface with a high level of accuracy. We show that both AKAP79 and S-AKAP84/D-AKAP1 bind RIIalpha very well (apparent K(D) values of 0.5 and 2 nM, respectively). Both proteins also bind RIIbeta quite well, but with three- to fourfold lower affinities than those observed versus RIIalpha. However, only S-AKAP84/D-AKAP1 interacts with RIalpha at a nanomolar affinity (apparent K(D) of 185 nM). In comparison, AKAP95 binds RIIalpha (apparent K(D) of 5.9 nM) with a tenfold higher affinity than RIIbeta and has no detectable binding to RIalpha. Surface competition assays with increasing concentrations of a competitor peptide covering amino acid residues 493 to 515 of the thyroid anchoring protein Ht31, demonstrated that Ht31, but not a proline-substituted peptide, Ht31-P, competed binding of RIIalpha and RIIbeta to all the AKAPs examined (EC(50)-values from 6 to 360 nM). Furthermore, RIalpha interaction with S-AKAP84/D-AKAP1 was competed (EC(50) 355 nM) with the same peptide. Here we report for the first time an approach to determine apparent rate- and equilibria binding constants for the interaction of all PKA isoforms with any AKAP as well as a novel approach for characterizing peptide competitors that disrupt PKA-AKAP anchoring.

  13. Preparative electrophoretic method for the purification of a hydrophobic membrane protein: subunit c of the mitochondrial ATP synthase from rat liver.

    PubMed

    Hagopian, K

    1999-09-10

    A method is described for the purification of subunit c of ATP synthase from rat liver mitochondria. After sample preparation and solvent extraction, the protein was purified to homogeneity by a single-step preparative electrophoretic procedure, using aqueous buffer and containing lithium dodecyl sulfate. The subunit is an extremely hydrophobic and insoluble protein and all solubilization attempts, using a variety of detergents, were unsuccessful except for lithium dodecyl sulfate. Buffer exchange and FPLC gel filtration removed the detergent from the purified sample, leaving the protein in a soluble form. The mammalian protein is composed of 75 amino acid residues, with a molecular mass of 7602 Da and is classified as a proteolipid. Subunit c accounts for 25 and 85% of the intralysosomal accumulation, within neurons, of storage material in juvenile and late-infantile forms of Batten's disease, respectively. This purification procedure allows access to a continuous supply of pure subunit c from a conventional source such as rat liver and preserves precious autopsy materials. The protein could be used as substrate in future proteolytic studies involving pepstatin-insensitive lysosomal proteases and for raising of more specific antibodies. The procedure could also be adapted/modified and used as a model for purifying other extremely insoluble proteins.

  14. Ref2, a regulatory subunit of the yeast protein phosphatase 1, is a novel component of cation homoeostasis.

    PubMed

    Ferrer-Dalmau, Jofre; González, Asier; Platara, Maria; Navarrete, Clara; Martínez, José L; Barreto, Lina; Ramos, José; Ariño, Joaquín; Casamayor, Antonio

    2010-02-24

    Maintenance of cation homoeostasis is a key process for any living organism. Specific mutations in Glc7, the essential catalytic subunit of yeast protein phosphatase 1, result in salt and alkaline pH sensitivity, suggesting a role for this protein in cation homoeostasis. We screened a collection of Glc7 regulatory subunit mutants for altered tolerance to diverse cations (sodium, lithium and calcium) and alkaline pH. Among 18 candidates, only deletion of REF2 (RNA end formation 2) yielded increased sensitivity to these conditions, as well as to diverse organic toxic cations. The Ref2F374A mutation, which renders it unable to bind Glc7, did not rescue the salt-related phenotypes of the ref2 strain, suggesting that Ref2 function in cation homoeostasis is mediated by Glc7. The ref2 deletion mutant displays a marked decrease in lithium efflux, which can be explained by the inability of these cells to fully induce the Na+-ATPase ENA1 gene. The effect of lack of Ref2 is additive to that of blockage of the calcineurin pathway and might disrupt multiple mechanisms controlling ENA1 expression. ref2 cells display a striking defect in vacuolar morphogenesis, which probably accounts for the increased calcium levels observed under standard growth conditions and the strong calcium sensitivity of this mutant. Remarkably, the evidence collected indicates that the role of Ref2 in cation homoeostasis may be unrelated to its previously identified function in the formation of mRNA via the APT (for associated with Pta1) complex.

  15. Tandem truncated rotavirus VP8* subunit protein with T cell epitope as non-replicating parenteral vaccine is highly immunogenic

    PubMed Central

    Wen, Xiaobo; Cao, Dianjun; Jones, Ronald W; Hoshino, Yasutaka; Yuan, Lijuan

    2015-01-01

    The two currently available live oral rotavirus vaccines, Rotarix® and RotaTeq®, are highly efficacious in the developed countries. However, the efficacy of such vaccines in resource deprived countries in Africa and Southeast Asia is low. We reported previously that a bacterially-expressed rotavirus P2-P[8] ΔVP8* subunit vaccine candidate administered intramuscularly elicited high-titers of neutralizing antibodies in guinea pigs and mice and significantly shortened the duration of diarrhea in neonatal gnotobiotic pigs upon oral challenge with virulent human rotavirus Wa strain. To further improve its vaccine potential and provide wider coverage against rotavirus strains of global and regional epidemiologic importance, we constructed 2 tandem recombinant VP8* proteins, P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8*-P[6] ΔVP8* based on Escherichia coli expression system. The two resulting recombinant tandem proteins were highly soluble and P2-P[8] ΔVP8*-P[8] ΔVP8* was generated with high yield. Moreover, guinea pigs immunized intramuscularly by 3 doses of the P2-P[8] ΔVP8*-P[8] ΔVP8* or P2-P[8] ΔVP8*-P[6] ΔVP8* vaccine with aluminum phosphate adjuvant developed high titers of homotypic and heterotypic neutralizing antibodies against human rotaviruses bearing G1-G4, G8, G9 and G12 with P[8], P[4] or P[6] combination. The results suggest that these 2 subunit vaccines in monovalent or bivalent formulation can provide antigenic coverage to almost all the rotavirus G (VP7) types and major P (VP4) types of global as well as regional epidemiologic importance. PMID:26091081

  16. Interaction of organic solvents with protein structures at protein-solvent interface.

    PubMed

    Khabiri, Morteza; Minofar, Babak; Brezovský, Jan; Damborský, Jiří; Ettrich, Rudiger

    2013-11-01

    The effect of non-denaturing concentrations of three different organic solvents, formamide, acetone and isopropanol, on the structure of haloalkane dehalogenases DhaA, LinB, and DbjA at the protein-solvent interface was studied using molecular dynamics simulations. Analysis of B-factors revealed that the presence of a given organic solvent mainly affects the dynamical behavior of the specificity-determining cap domain, with the exception of DbjA in acetone. Orientation of organic solvent molecules on the protein surface during the simulations was clearly dependent on their interaction with hydrophobic or hydrophilic surface patches, and the simulations suggest that the behavior of studied organic solvents in the vicinity of hyrophobic patches on the surface is similar to the air/water interface. DbjA was the only dimeric enzyme among studied haloalkane dehalogenases and provided an opportunity to explore effects of organic solvents on the quaternary structure. Penetration and trapping of organic solvents in the network of interactions between both monomers depends on the physico-chemical properties of the organic solvents. Consequently, both monomers of this enzyme oscillate differently in different organic solvents. With the exception of LinB in acetone, the structures of studied enzymes were stabilized in water-miscible organic solvents.

  17. Crystallization of the glycogen-binding domain of the AMP-activated protein kinase β subunit and preliminary X-ray analysis

    SciTech Connect

    Polekhina, Galina Feil, Susanne C.; Gupta, Abhilasha; O’Donnell, Paul; Stapleton, David; Parker, Michael W.

    2005-01-01

    The glycogen-binding domain of the AMP-activated kinase β subunit has been crystallized in the presence of β-cyclodextrin. The structure has been determined by single isomorphous replacement and threefold averaging using in-house X-ray data collected from selenomethionine-substituted protein. AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolism in response to energy demand and supply by adjusting the ATP-generating and ATP-consuming pathways. AMPK potentially plays a critical role in diabetes and obesity as it is known to be activated by metforin and rosiglitazone, drugs used for the treatment of type II diabetes. AMPK is a heterotrimer composed of a catalytic α subunit and two regulatory subunits, β and γ. Mutations in the γ subunit are known to cause glycogen accumulation, leading to cardiac arrhythmias. Recently, a functional glycogen-binding domain (GBD) has been identified in the β subunit. Here, the crystallization of GBD in the presence of β-cyclodextrin is reported together with preliminary X-ray data analysis allowing the determination of the structure by single isomorphous replacement and threefold averaging using in-house X-ray data collected from a selenomethionine-substituted protein.

  18. Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo

    PubMed Central

    Lim, Sung In; Hahn, Young S.; Kwon, Inchan

    2015-01-01

    Albumin fusion/conjugation (albumination) has been an effective method to prolong in vivo half-life of therapeutic proteins. However, its broader application to proteins with complex folding pathway or multi-subunit is restricted by incorrect folding, poor expression, heterogeneity, and loss of native activity of the proteins linked to albumin. We hypothesized that the site-specific conjugation of albumin to a permissive site of a target protein will expand the utilities of albumin as a therapeutic activity extender to proteins with a complex structure. We show here the genetic incorporation of a non-natural amino acid (NNAA) followed by chemoselective albumin conjugation to prolong therapeutic activity in vivo. Urate oxidase (Uox), a therapeutic enzyme for treatment of hyperuricemia, is a homotetramer with multiple surface lysines, limiting conventional approaches for albumination. Incorporation of p-azido-l-phenylalanine into two predetermined positions of Uox allowed site-specific linkage of dibenzocyclooctyne-derivatized human serum albumin (HSA) through strain-promoted azide-alkyne cycloaddition (SPAAC). The bio-orthogonality of SPAAC resulted in the production of a chemically well-defined conjugate, Uox-HSA, with a retained enzymatic activity. Uox-HSA had a half-life of 8.8 h in mice, while wild-type Uox had a half-life of 1.3 h. The AUC increased 5.5-fold (1657 vs. 303 mU/mL × h). These results clearly demonstrated that site-specific albumination led to the prolonged enzymatic activity of Uox in vivo. Site-specific albumination enabled by NNAA incorporation and orthogonal chemistry demonstrates its promise for the development of long-acting protein therapeutics with high potency and safety. PMID:25862515

  19. Proteins at interfaces probed by chiral vibrational sum frequency generation spectroscopy.

    PubMed

    Yan, Elsa C Y; Wang, Zhuguang; Fu, Li

    2015-02-19

    Characterizations of protein structures at interfaces are important in solving an array of fundamental and engineering problems, including understanding transmembrane signal transduction and molecular transport processes and development of biomaterials to meet the needs of biomedical and energy research. However, in situ and real-time characterization of protein secondary structures is challenging because it requires physical methods that are selective to both interface and secondary structures. Here, we summarize recent experimental developments in our laboratory of chiral vibrational sum frequency generation spectroscopy (SFG) for analyzing protein structures at interfaces. We showed that chiral SFG provides vibrational optical signatures of the peptide N-H stretch and amide I modes that can distinguish various protein secondary structures. Using these signatures, we further applied chiral SFG to probe orientations and folding kinetics of proteins at interfaces. Our results show that chiral SFG is a background-free, label-free, in situ, and real-time vibrational method for studying proteins at interfaces. This recent progress demonstrates the potential of chiral SFG in solving problems related to proteins and other chiral biopolymers at interfaces.

  20. BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

    PubMed

    Wei, Qing; La, David; Kihara, Daisuke

    2017-01-01

    Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

  1. Molecular characterization of iron binding proteins, transferrin and ferritin heavy chain subunit, from the bumblebee Bombus ignitus.

    PubMed

    Wang, Dong; Kim, Bo Yeon; Lee, Kwang Sik; Yoon, Hyung Joo; Cui, Zheng; Lu, Wei; Jia, Jing Ming; Kim, Doh Hoon; Sohn, Hung Dae; Jin, Byung Rae

    2009-01-01

    Transferrin and ferritin are iron-binding proteins involved in transport and storage of iron as part of iron metabolism. Here, we describe the cDNA cloning and characterization of transferrin (Bi-Tf) and the ferritin heavy chain subunit (Bi-FerHCH), from the bumblebee Bombus ignitus. Bi-Tf cDNA spans 2340 bp and encodes a protein of 706 amino acids and Bi-FerHCH cDNA spans 1393 bp and encodes a protein of 217 amino acids. Comparative analysis revealed that Bi-Tf appears to have residues comprising iron-binding sites in the N-terminal lobe, and Bi-FerHCH contains a 5'UTR iron-responsive element and seven conserved amino acid residues associated with a ferroxidase center. The Bi-Tf and Bi-FerHCH cDNAs were expressed as 79 kDa and 27 kDa polypeptides, respectively, in baculovirus-infected insect Sf9 cells. Northern blot analysis revealed that Bi-Tf exhibits fat body-specific expression and Bi-FerHCH shows ubiquitous expression. The expression profiles of the Bi-Tf and Bi-FerHCH in the fat body of B. ignitus worker bees revealed that Bi-Tf and Bi-FerHCH are differentially induced in a time-dependent manner in a single insect by wounding, bacterial challenge, and iron overload.

  2. Targeting the binding interface on a shared receptor subunit of a cytokine family enables the inhibition of multiple member cytokines with selectable target spectrum.

    PubMed

    Nata, Toshie; Basheer, Asjad; Cocchi, Fiorenza; van Besien, Richard; Massoud, Raya; Jacobson, Steven; Azimi, Nazli; Tagaya, Yutaka

    2015-09-11

    The common γ molecule (γc) is a shared signaling receptor subunit used by six γc-cytokines. These cytokines play crucial roles in the differentiation of the mature immune system and are involved in many human diseases. Moreover, recent studies suggest that multiple γc-cytokines are pathogenically involved in a single disease, thus making the shared γc-molecule a logical target for therapeutic intervention. However, the current therapeutic strategies seem to lack options to treat such cases, partly because of the lack of appropriate neutralizing antibodies recognizing the γc and, more importantly, because of the inherent and practical limitations in the use of monoclonal antibodies. By targeting the binding interface of the γc and cytokines, we successfully designed peptides that not only inhibit multiple γc-cytokines but with a selectable target spectrum. Notably, the lead peptide inhibited three γc-cytokines without affecting the other three or non-γc-cytokines. Biological and mutational analyses of our peptide provide new insights to our current understanding on the structural aspect of the binding of γc-cytokines the γc-molecule. Furthermore, we provide evidence that our peptide, when conjugated to polyethylene glycol to gain stability in vivo, efficiently blocks the action of one of the target cytokines in animal models. Collectively, our technology can be expanded to target various combinations of γc-cytokines and thereby will provide a novel strategy to the current anti-cytokine therapies against immune, inflammatory, and malignant diseases.

  3. A dominant conformational role for amino acid diversity in minimalist protein–protein interfaces

    SciTech Connect

    Gilbreth, Ryan N.; Esaki, Kaori; Koide, Akiko; Sidhu, Sachdev S.; Koide, Shohei

    2008-08-01

    Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed “monobodies.” One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose-binding protein. The YSX monobody bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution X-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces.

  4. Flexibility and small pockets at protein–protein interfaces: New insights into druggability

    PubMed Central

    Jubb, Harry; Blundell, Tom L.; Ascher, David B.

    2015-01-01

    The transient assembly of multiprotein complexes mediates many aspects of cell regulation and signalling in living organisms. Modulation of the formation of these complexes through targeting protein–protein interfaces can offer greater selectivity than the inhibition of protein kinases, proteases or other post-translational regulatory enzymes using substrate, co-factor or transition state mimetics. However, capitalising on protein–protein interaction interfaces as drug targets has been hindered by the nature of interfaces that tend to offer binding sites lacking the well-defined large cavities of classical drug targets. In this review we posit that interfaces formed by concerted folding and binding (disorder-to-order transitions on binding) of one partner and other examples of interfaces where a protein partner is bound through a continuous epitope from a surface-exposed helix, flexible loop or chain extension may be more tractable for the development of “orthosteric”, competitive chemical modulators; these interfaces tend to offer small-volume but deep pockets and/or larger grooves that may be bound tightly by small chemical entities. We discuss examples of such protein–protein interaction interfaces for which successful chemical modulators are being developed. PMID:25662442

  5. A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli

    PubMed Central

    Smith, Corey L.; Matheson, Timothy D.; Trombly, Daniel J.; Sun, Xiaoming; Campeau, Eric; Han, Xuemei; Yates, John R.; Kaufman, Paul D.

    2014-01-01

    Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element–containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. In addition, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces α-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus. PMID:25057015

  6. The primary structure of component 8c-1, a subunit protein of intermediate filaments in wool keratin. Relationships with proteins from other intermediate filaments.

    PubMed Central

    Dowling, L M; Crewther, W G; Inglis, A S

    1986-01-01

    Component 8c-1, one of four highly homologous component-8 subunit proteins present in the microfibrils of wool, was isolated as its S-carboxymethyl derivative and its amino acid sequence was determined. Large peptides were isolated after cleaving the protein chemically or enzymically and the sequence of each was determined with an automatic Sequenator. The peptides were ordered by sequence overlaps and, in some instances, by homology with known sequences from other component-8 subunits. The C-terminal residues were identified by three procedures. Full details of the various procedures used have been deposited as Supplementary Publication SUP 50133 (4 pp.) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5. The result showed that the protein comprises 412 residues and has an Mr, including the N-terminal acetyl group, of 48,300. The sequence of residues 98-200 of component 8c-1 was found to correspond to the partial or complete sequences of four homologous type I helical segments previously isolated from helical fragments recovered from chymotryptic digests of microfibrillar proteins of wool [Crewther & Dowling (1971) Appl. Polym. Symp. 18, 1-20; Crewther, Gough, Inglis & McKern (1978) Text. Res. J. 48, 160-162; Gough, Inglis & Crewther (1978) Biochem. J. 173, 385]. Considered in relation to amino acid sequences of other intermediate-filament proteins, the sequence is in accord with the view that keratin filament proteins are of two types [Hanukoglu & Fuchs (1983) Cell (Cambridge, Mass.) 33, 915-924]. Filament proteins from non-keratinous tissues, such as desmin, vimentin, neurofilament proteins and the glial fibrillary acidic protein, which form monocomponent filaments, constitute a third type. It is suggested that as a whole the proteins from intermediate filaments be classed as filamentins, the three types at present identified forming

  7. Nonstructural protein 3 of hepatitis C virus blocks the distribution of the free catalytic subunit of cyclic AMP-dependent protein kinase.

    PubMed Central

    Borowski, P; Oehlmann, K; Heiland, M; Laufs, R

    1997-01-01

    Chronic hepatitis resulting from hepatitis C virus (HCV) infection develops into cirrhosis in at least half of infected patients and increases the risk of hepatocellular carcinoma. The pathogenic effects of a number of viruses result from the disturbance of intracellular signal cascades caused by viral antigens. Therefore, we investigated the interaction of nonstructural protein 3 (NS3) of HCV with the cyclic AMP-dependent signal pathway. We found a similarity between the HCV sequence Arg-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg localized in NS3 and the general consensus sequence of protein kinase A (PKA). Consequently, the catalytic (C) subunit of PKA bound to a bacterially expressed fragment of HCV polyprotein containing amino acid residues 1189 to 1525. When this fragment was introduced into cells, it inhibited the translocation of the C subunit into the nucleus after stimulation with forskolin. The result of this inhibition was significantly reduced histone phosphorylation. Therefore, the presence of NS3 in the cytoplasm of infected cells may affect a wide range of PKA functions and contribute to the pathogenesis of the diseases caused by HCV. PMID:9060639

  8. TIM29 is a subunit of the human carrier translocase required for protein transport.

    PubMed

    Callegari, Sylvie; Richter, Frank; Chojnacka, Katarzyna; Jans, Daniel C; Lorenzi, Isotta; Pacheu-Grau, David; Jakobs, Stefan; Lenz, Christof; Urlaub, Henning; Dudek, Jan; Chacinska, Agnieszka; Rehling, Peter

    2016-12-01

    Hydrophobic inner mitochondrial membrane proteins with internal targeting signals, such as the metabolite carriers, use the carrier translocase (TIM22 complex) for transport into the inner membrane. Defects in this transport pathway have been associated with neurodegenerative disorders. While the TIM22 complex is well studied in baker's yeast, very little is known about the mammalian TIM22 complex. Using immunoprecipitation, we purified the human carrier translocase and identified a mitochondrial inner membrane protein TIM29 as a novel component, specific to metazoa. We show that TIM29 is a constituent of the 440 kDa TIM22 complex and interacts with oxidized TIM22. Our analyses demonstrate that TIM29 is required for the structural integrity of the TIM22 complex and for import of substrate proteins by the carrier translocase.

  9. Computer simulations and neutron reflectivity of proteins at interfaces.

    PubMed

    Mungikar, Amol A; Forciniti, Daniel

    2002-12-16

    Computer simulations in conjunction with neutron reflectivity is an excellent combination for the study of biological materials at solid-liquid interfaces: Both techniques have excellent resolution levels (Angströms) and they are mature. A stronger interaction between physicists and biologists will allow the use of these two approaches in topics of biological-biomedical interest.

  10. Molecular recognition at the dimer interface of a class mu glutathione transferase: role of a hydrophobic interaction motif in dimer stability and protein function.

    PubMed

    Hornby, Judith A T; Codreanu, Simona G; Armstrong, Richard N; Dirr, Heini W

    2002-12-03

    Cytosolic glutathione (GSH) transferases (GSTs) exist as stable homo- and heterodimers. Interactions at the subunit interface serve an important role in stabilizing the subunit tertiary structures of all GSH transferases. In addition, the dimer is required to maintain functional conformations at the active site on each subunit and the nonsubstrate ligand binding site at the dimer interface [Dirr, H. W. (2001) Chem.-Biol. Interact. 133, 19-23]. In this study, we report on the contribution of a specific intersubunit hydrophobic motif in rGSTM1-1 to dimer stability and protein function. The motif consists of the side chain of F56 from one subunit intercalated between helices 4 and 5 of the second subunit. Replacement of F56 with the hydrophilic side chains of serine, arginine, and glutamate results in a change in the structure of the active site, a marked diminution in catalytic efficiency, and alterations in the ability to bind nonsubstrate ligands. The mutations also affect the ability of the enzyme to bind GSH and the substrate analogue glutathione sulfonate. The functionality of rGSTM1-1 was disrupted to the greatest extent for the F56E mutant. Though mutations at this position do not alter the three-state equilibrium folding process for rGSTM1-1 (i.e., N(2) <--> 2I <--> 2U), destabilizing mutations at position 56 shift the equilibrium between the folded dimer (N(2)) and the monomeric intermediate (I) toward the latter conformational state. The transition to the unfolded state (U) is not significantly affected. The folded monomeric intermediate is also observed by electrospray ionization mass spectrometry. The amount of the intermediate is dependent on protein concentration and the residue at position 56. Mutations at position 56 have little impact on the secondary structure and stability of the monomeric folding intermediate. The dimerization process is proposed to induce a conformational change in the loop containing F56, resulting in improved stability and

  11. The CAD-score web server: contact area-based comparison of structures and interfaces of proteins, nucleic acids and their complexes.

    PubMed

    Olechnovič, Kliment; Venclovas, Ceslovas

    2014-07-01

    The Contact Area Difference score (CAD-score) web server provides a universal framework to compute and analyze discrepancies between different 3D structures of the same biological macromolecule or complex. The server accepts both single-subunit and multi-subunit structures and can handle all the major types of macromolecules (proteins, RNA, DNA and their complexes). It can perform numerical comparison of both structures and interfaces. In addition to entire structures and interfaces, the server can assess user-defined subsets. The CAD-score server performs both global and local numerical evaluations of structural differences between structures or interfaces. The results can be explored interactively using sortable tables of global scores, profiles of local errors, superimposed contact maps and 3D structure visualization. The web server could be used for tasks such as comparison of models with the native (reference) structure, comparison of X-ray structures of the same macromolecule obtained in different states (e.g. with and without a bound ligand), analysis of nuclear magnetic resonance (NMR) structural ensemble or structures obtained in the course of molecular dynamics simulation. The web server is freely accessible at: http://www.ibt.lt/bioinformatics/cad-score.

  12. Labeled Protein Recognition at a Membrane Bilayer Interface by Embedded Synthetic Receptors

    PubMed Central

    2015-01-01

    Self-folding deep cavitands embedded in a supported lipid bilayer are capable of recognizing suitably labeled proteins at the bilayer interface. The addition of a choline derived binding “handle” to a number of different proteins allows their selective noncovalent recognition, with association constants on the order of 105 M–1. The proteins are displayed at the water:bilayer interface, and a single binding handle allows recognition of the large, charged protein by a small molecule synthetic receptor via complementary shape and charge interactions. PMID:25130415

  13. Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

    PubMed

    Duffy, Fergal J; O'Donovan, Darragh; Devocelle, Marc; Moran, Niamh; O'Connell, David J; Shields, Denis C

    2015-03-23

    Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.

  14. Nicotinic pharmacophore: the pyridine N of nicotine and carbonyl of acetylcholine hydrogen bond across a subunit interface to a backbone NH.

    PubMed

    Blum, Angela P; Lester, Henry A; Dougherty, Dennis A

    2010-07-27

    Pharmacophore models for nicotinic agonists have been proposed for four decades. Central to these models is the presence of a cationic nitrogen and a hydrogen bond acceptor. It is now well-established that the cationic center makes an important cation-pi interaction to a conserved tryptophan, but the donor to the proposed hydrogen bond acceptor has been more challenging to identify. A structure of nicotine bound to the acetylcholine binding protein predicted that the binding partner of the pharmacophore's second component was a water molecule, which also hydrogen bonds to the backbone of the complementary subunit of the receptors. Here we use unnatural amino acid mutagenesis coupled with agonist analogs to examine whether such a hydrogen bond is functionally significant in the alpha4beta2 neuronal nAChR, the receptor most associated with nicotine addiction. We find evidence for the hydrogen bond with the agonists nicotine, acetylcholine, carbamylcholine, and epibatidine. These data represent a completed nicotinic pharmacophore and offer insight into the design of new therapeutic agents that selectively target these receptors.

  15. Evidence That Bacteriophage λ Kil Peptide Inhibits Bacterial Cell Division by Disrupting FtsZ Protofilaments and Sequestering Protein Subunits.

    PubMed

    Hernández-Rocamora, Víctor M; Alfonso, Carlos; Margolin, William; Zorrilla, Silvia; Rivas, Germán

    2015-08-14

    The effects of Kil peptide from bacteriophage λ on the assembly of Escherichia coli FtsZ into one subunit thick protofilaments were studied using combined biophysical and biochemical methods. Kil peptide has recently been identified as the factor from bacteriophage λ responsible for the inhibition of bacterial cell division during lytic cycle, targeting FtsZ polymerization. Here, we show that this antagonist blocks FtsZ assembly into GTP-dependent protofilaments, producing a wide distribution of smaller oligomers compared with the average size of the intact protofilaments. The shortening of FtsZ protofilaments by Kil is detectable at concentrations of the peptide in the low micromolar range, the mid-point of the inhibition being close to its apparent affinity for GDP-bound FtsZ. This antagonist not only interferes with FtsZ assembly but also reverses the polymerization reaction. The negative regulation by Kil significantly reduces the GTPase activity of FtsZ protofilaments, and FtsZ polymers assembled in guanosine-5'-[(α,β)-methyleno]triphosphate are considerably less sensitive to Kil. Our results suggest that, at high concentrations, Kil may use an inhibition mechanism involving the sequestration of FtsZ subunits, similar to that described for other inhibitors like the SOS response protein SulA or the moonlighting enzyme OpgH. This mechanism is different from those employed by the division site selection antagonists MinC and SlmA. This work provides new insight into the inhibition of FtsZ assembly by phages, considered potential tools against bacterial infection.

  16. The atypical two-subunit σ factor from Bacillus subtilis is regulated by an integral membrane protein and acid stress.

    PubMed

    Davis, Maria C; Smith, Logan K; MacLellan, Shawn R

    2016-02-01

    Extracytoplasmic function (ECF) σ factors constitute a major component of the physicochemical sensory apparatus in bacteria. Most ECF σ factors are co-expressed with a negative regulator called an anti-σ factor that binds to its cognate σ factor and sequesters it from productive association with core RNA polymerase (RNAP). Anti-σ factors constitute an important element of signal transduction pathways that mediate an appropriate transcriptional response to changing environmental conditions. The Bacillus subtilis genome encodes seven canonical ECF σ factors and six of these are co-expressed with experimentally verified anti-σ factors. B. subtilis also expresses an ECF-like atypical two-subunit σ factor composed of subunits SigO and RsoA that becomes active after exposure to certain cell-wall-acting antibiotics and to growth under acidic conditions. This work describes the identification and preliminary characterization of a protein (RsiO, formerly YvrL) that constitutes the anti-σ factor cognate to SigO-RsoA. Synthesis of RsiO represses SigO-RsoA-dependent transcription initiation by binding the N-terminus of SigO under neutral (pH 7) conditions. Reconstitution of the SigO-RsoA-RsiO regulatory system into a heterologous host reveals that the imposition of acid stress (pH 5.4) abolishes the ability of RsiO to repress SigO-RsoA-dependent transcription and this correlates with loss of RsiO binding affinity for SigO. A current model for RsiO function indicates that RsiO responds, either directly or indirectly, to increased extracytoplasmic hydrogen ion concentration and becomes inactivated. This results in the release of SigO into the cytoplasm, where it productively associates with RsoA and core RNAP to initiate transcription from target promoters in the cell.

  17. Assemblies of glutamate receptor subunits with post-synaptic density proteins and their alterations in Parkinson's disease.

    PubMed

    Gardoni, Fabrizio; Ghiglieri, Veronica; Di Luca, Monica; Calabresi, Paolo

    2010-01-01

    N-methyl-D-aspartate (NMDA) receptors have been implicated as a mediator of neuronal injury associated with many neurological disorders including ischemia, epilepsy, brain trauma, dementia and neurodegenerative disorders such as Parkinson's disease (PD) and Alzheimer's disease. To this, non-selective NMDA receptor antagonists have been tried and have been shown to be effective in many experimental animal models of disease, and some of these compounds have moved into clinical trials. However, the initial enthusiasm for this approach has waned, because the therapeutic index for most NMDA antagonists is quite poor, with significant adverse effects at clinically effective doses, thus limiting their utility. More recently, the concept that the exact pathways downstream NMDA receptor activation could represent a key variable element among neurological disorders has been put forward. In particular, variations in NMDA receptor subunit composition could be important in different disorders, both in the pathophysiological mechanisms of cell death and in the application of specific symptomatic therapies. As to PD, NMDA receptor complex has been shown to be altered in experimental models of parkinsonism and in PD in humans. Further, it has become increasingly evident that the NMDA receptor complex is intimately involved in the regulation of corticostriatal long-term potentiation, which is altered in experimental parkinsonism. The following sections will examine the modifications of specific NMDA receptor subunits as well as post-synaptic associated signalling complex including kinases and scaffolding proteins in experimental parkinsonism. These findings may allow the identification of specific molecular targets whose pharmacological or genetic manipulation might lead to innovative therapies for PD.

  18. Molecular cloning, expression and functional analysis of three subunits of protein phosphatase 2A (PP2A) from black tiger shrimps (Penaeus monodon).

    PubMed

    Zhao, Chao; Wang, Yan; Fu, Mingjun; Yang, Keng; Qiu, Lihua

    2017-02-01

    Protein phosphatase 2A (PP2A) is a cellular serine-threonine (Ser/Thr) phosphatase that plays a crucial role in regulating most cellular functions. In the present study, the full-length cDNAs of three subunits of PmPP2A (PmPP2A-A, PP2A-B and PP2A-C) were cloned from Penaeus monodon, which are the first available for shrimps. Sequence analysis showed that PmPP2A-A, PmPP2A-B and PmPP2A-C encoded polypeptides of 591, 443, and 324 amino acids, respectively. The mRNAs of three subunits of PmPP2A were expressed constitutively in all tissues examined, and predominantly in the ovaries. In ovarian maturation stages, the three subunits of PmPP2A were continuously but differentially expressed. Dopamine and 5-hydroxytryptamine injection experiments were conducted to study the expression profile of three subunits of PmPP2A, and the results indicated that PmPP2A played a negative regulatory role in the process of ovarian maturation. In addition, the recombinant proteins of three subunits of PmPP2A were successfully obtained, and the phosphatase activity of PmPP2A was tested in vitro. The results of this study will advance our understanding about the molecular mechanisms of PmPP2A in Penaeus monodon.

  19. The dipeptidyl-aminopeptidase-like protein 6 is an integral voltage sensor-interacting beta-subunit of neuronal K(V)4.2 channels.

    PubMed

    Dougherty, Kevin; Tu, Liwei; Deutsch, Carol; Covarrubias, Manuel

    2009-01-01

    Auxiliary beta-subunits dictate the physiological properties of voltage-gated K(+) (K(V)) channels in excitable tissues. In many instances, however, the underlying mechanisms of action are poorly understood. The dipeptidyl-aminopeptidase-like protein 6 (DPP6) is a specific beta-subunit of neuronal K(V)4 channels, which may promote gating through interactions between the single transmembrane segment of DPP6 and the channel's voltage sensing domain (VSD). A combination of gating current measurements and protein biochemistry (in-vitro translation and co-immunoprecipitations) revealed preferential physical interaction between the isolated K(V)4.2-VSD and DPP6. Significantly weaker interactions were detected between DPP6 and K(V)1.3 channels or the K(V)4.2 pore domain. More efficient gating charge movement resulting from a direct interaction between DPP6 and the K(V)4.2-VSD is unique among the known actions of K(V) channel beta-subunits. This study shows that the modular VSD of a K(V) channel can be directly regulated by transmembrane protein-protein interactions involving an extrinsic beta-subunit. Understanding these interactions may shed light on the pathophysiology of recently identified human disorders associated with mutations affecting the dpp6 gene.

  20. The eukaryote-specific N-terminal extension of ribosomal protein S31 contributes to the assembly and function of 40S ribosomal subunits

    PubMed Central

    Fernández-Pevida, Antonio; Martín-Villanueva, Sara; Murat, Guillaume; Lacombe, Thierry; Kressler, Dieter; de la Cruz, Jesús

    2016-01-01

    The archaea-/eukaryote-specific 40S-ribosomal-subunit protein S31 is expressed as an ubiquitin fusion protein in eukaryotes and consists of a conserved body and a eukaryote-specific N-terminal extension. In yeast, S31 is a practically essential protein, which is required for cytoplasmic 20S pre-rRNA maturation. Here, we have studied the role of the N-terminal extension of the yeast S31 protein. We show that deletion of this extension partially impairs cell growth and 40S subunit biogenesis and confers hypersensitivity to aminoglycoside antibiotics. Moreover, the extension harbours a nuclear localization signal that promotes active nuclear import of S31, which associates with pre-ribosomal particles in the nucleus. In the absence of the extension, truncated S31 inefficiently assembles into pre-40S particles and two subpopulations of mature small subunits, one lacking and another one containing truncated S31, can be identified. Plasmid-driven overexpression of truncated S31 partially suppresses the growth and ribosome biogenesis defects but, conversely, slightly enhances the hypersensitivity to aminoglycosides. Altogether, these results indicate that the N-terminal extension facilitates the assembly of S31 into pre-40S particles and contributes to the optimal translational activity of mature 40S subunits but has only a minor role in cytoplasmic cleavage of 20S pre-rRNA at site D. PMID:27422873

  1. Rice G-protein subunits qPE9-1 and RGB1 play distinct roles in abscisic acid responses and drought adaptation.

    PubMed

    Zhang, Dong-Ping; Zhou, Yong; Yin, Jian-Feng; Yan, Xue-Jiao; Lin, Sheng; Xu, Wei-Feng; Baluška, František; Wang, Yi-Ping; Xia, Yi-Ji; Liang, Guo-hua; Liang, Jian-Sheng

    2015-10-01

    Heterotrimeric GTP-binding protein (G-protein)-mediated abscisic acid (ABA) and drought-stress responses have been documented in numerous plant species. However, our understanding of the function of rice G-protein subunits in ABA signalling and drought tolerance is limited. In this study, the function of G-protein subunits in ABA response and drought resistance in rice plants was explored. It was found that the transcription level of qPE9-1 (rice Gγ subunit) gradually decreased with increasing ABA concentration and the lack of qPE9-1 showed an enhanced drought tolerance in rice plants. In contrast, mRNA levels of RGB1 (rice Gβ subunit) were significantly upregulated by ABA treatment and the lack of RGB1 led to reduced drought tolerance. Furthermore, the results suggested that qPE9-1 negatively regulates the ABA response by suppressing the expression of key transcription factors involved in ABA and stress responses, while RGB1 positively regulates ABA biosynthesis by upregulating NCED gene expression under both normal and drought stress conditions. Taken together, it is proposed that RGB1 is a positive regulator of the ABA response and drought adaption in rice plants, whereas qPE9-1 is modulated by RGB1 and functions as a negative regulator in the ABA-dependent drought-stress responses.

  2. Cloning and targeted mutations of G alpha 7 and G alpha 8, two developmentally regulated G protein alpha-subunit genes in Dictyostelium.

    PubMed Central

    Wu, L; Gaskins, C; Zhou, K; Firtel, R A; Devreotes, P N

    1994-01-01

    GTP-binding protein (G protein)-mediated signal transduction pathways play essential roles during the aggregation and differentiation process of Dictyostelium. In addition to the five known G protein alpha-subunit genes, we recently identified three novel alpha-subunit genes, G alpha 6, G alpha 7, and G alpha 8, using the polymerase chain reaction technique. We present here a more complete analysis of G alpha 7 and G alpha 8. The cDNAs of these two genes were cloned, and their complete nucleotide sequences were determined. Sequence analyses indicate that G alpha 8 possesses some unusual features. It lacks the "TCATDT" motif, a sequence of amino acids highly conserved among G alpha subunits, and has an additional 50 amino acids at its C-terminus consisting of long stretches of asparagine. Moreover, G alpha 8 is unusually resistant to protease digestion, which may indicate a slow GTP hydrolysis rate. The possible functions of these alpha-subunits were assessed by generating mutants lacking G alpha 7 or G alpha 8 by gene targeting through homologous recombination and by overexpressing G alpha 7 or G alpha 8 protein. Overexpression of G alpha 7 resulted in abnormal morphogenesis starting at the slug stage, whereas analysis of the other strains failed to reveal any obvious growth or developmental defects under either normal or stressful conditions. The implications of these results are discussed. Images PMID:7949425

  3. Rescore protein-protein docked ensembles with an interface contact statistics.

    PubMed

    Mezei, Mihaly

    2017-02-01

    The recently developed statistical measure for the type of residue-residue contact at protein complex interfaces, based on a parameter-free definition of contact, has been used to define a contact score that is correlated with the likelihood of correctness of a proposed complex structure. Comparing the proposed contact scores on the native structure and on a set of model structures the proposed measure was shown to generally favor the native structure but in itself was not able to reliably score the native structure to be the best. Adjusting the scores of redocking experiments with the contact score showed that the adjusted score was able to move up the ranking of the native-like structure among the proposed complexes when the native-like was not ranked the best by the respective program. Tests on docking of unbound proteins compared the contact scores of the complexes with the contact score of the crystal structure again showing the tendency of the contact score to favor native-like conformations. The possibility of using the contact score to improve the determination of biological dimers in a crystal structure was also explored. Proteins 2017; 85:235-241. © 2016 Wiley Periodicals, Inc.

  4. Drosophila Condensin II subunit Chromosome-associated protein D3 regulates cell fate determination through non-cell-autonomous signaling

    PubMed Central

    Klebanow, Lindsey R.; Peshel, Emanuela C.; Schuster, Andrew T.; De, Kuntal; Sarvepalli, Kavitha; Lemieux, Madeleine E.; Lenoir, Jessica J.; Moore, Adrian W.; McDonald, Jocelyn A.

    2016-01-01

    The pattern of the Drosophila melanogaster adult wing is heavily influenced by the expression of proteins that dictate cell fate decisions between intervein and vein during development. dSRF (Blistered) expression in specific regions of the larval wing disc promotes intervein cell fate, whereas EGFR activity promotes vein cell fate. Here, we report that the chromatin-organizing protein CAP-D3 acts to dampen dSRF levels at the anterior/posterior boundary in the larval wing disc, promoting differentiation of cells into the anterior crossvein. CAP-D3 represses KNOT expression in cells immediately adjacent to the anterior/posterior boundary, thus blocking KNOT-mediated repression of EGFR activity and preventing cell death. Maintenance of EGFR activity in these cells depresses dSRF levels in the neighboring anterior crossvein progenitor cells, allowing them to differentiate into vein cells. These findings uncover a novel transcriptional regulatory network influencing Drosophila wing vein development, and are the first to identify a Condensin II subunit as an important regulator of EGFR activity and cell fate determination in vivo. PMID:27317808

  5. A Mitochondrial ATP synthase Subunit Interacts with TOR Signaling to Modulate Protein Homeostasis and Lifespan in Drosophila

    PubMed Central

    Sun, Xiaoping; Wheeler, Charles T.; Yolitz, Jason; Laslo, Mara; Alberico, Thomas; Sun, Yaning; Song, Qisheng; Zou, Sige

    2014-01-01

    SUMMARY Diet composition is a critical determinant of lifespan and nutrient imbalance is detrimental health. However, how nutrients interact with genetic factors to modulate lifespan remains elusive. We investigated how diet composition influences mitochondrial ATP synthase subunit d (ATPsyn-d) in modulating lifespan in Drosophila. ATPsyn-d knockdown extended lifespan in females fed low carbohydrate-to-protein (C:P) diets, but not the high C:P ratio diet. This extension was associated with increased resistance to oxidative stress, transcriptional changes in metabolism, proteostasis and immune genes, reduced protein damage and aggregation, and reduced phosphorylation of S6K and ERK in TOR and MAPK signaling, respectively. ATPsyn-d knockdown did not extend lifespan in females with reduced TOR signaling induced genetically by Tsc2 overexpression or pharmacologically by rapamycin. Our data reveal a link among diet, mitochondria, MAPK and TOR signaling in aging and stresses the importance of considering genetic background and diet composition in implementing interventions for promoting healthy aging. PMID:25220459

  6. Listerin-dependent nascent protein ubiquitination relies on ribosome subunit dissociation.

    PubMed

    Shao, Sichen; von der Malsburg, Karina; Hegde, Ramanujan S

    2013-06-06

    Quality control of defective mRNAs relies on their translation to detect the lesion. Aberrant proteins are therefore an obligate byproduct of mRNA surveillance and must be degraded to avoid disrupting protein homeostasis. These defective translation products are thought to be ubiquitinated at the ribosome, but the mechanism of ubiquitin ligase selectivity for these ribosomes is not clear. Here, we in vitro reconstitute ubiquitination of nascent proteins produced from aberrant mRNAs. Stalled 80S ribosome-nascent chain complexes are dissociated by the ribosome recycling factors Hbs1/Pelota/ABCE1 to a unique 60S-nascent chain-tRNA complex. The ubiquitin ligase Listerin preferentially recognizes 60S-nascent chains and triggers efficient nascent chain ubiquitination. Interfering with Hbs1 function stabilizes 80S complexes, precludes efficient Listerin recruitment, and reduces nascent chain ubiquitination. Thus, ribosome recycling factors control Listerin localization, explaining how translation products of mRNA surveillance are efficiently ubiquitinated while sparing translating ribosomes.

  7. Sequence Analysis and Comparative Study of the Protein Subunits of Archaeal RNase P

    PubMed Central

    Samanta, Manoj P.; Lai, Stella M.; Daniels, Charles J.; Gopalan, Venkat

    2016-01-01

    RNase P, a ribozyme-based ribonucleoprotein (RNP) complex that catalyzes tRNA 5′-maturation, is ubiquitous in all domains of life, but the evolution of its protein components (RNase P proteins, RPPs) is not well understood. Archaeal RPPs may provide clues on how the complex evolved from an ancient ribozyme to an RNP with multiple archaeal and eukaryotic (homologous) RPPs, which are unrelated to the single bacterial RPP. Here, we analyzed the sequence and structure of archaeal RPPs from over 600 available genomes. All five RPPs are found in eight archaeal phyla, suggesting that these RPPs arose early in archaeal evolutionary history. The putative ancestral genomic loci of archaeal RPPs include genes encoding several members of ribosome, exosome, and proteasome complexes, which may indicate coevolution/coordinate regulation of RNase P with other core cellular machineries. Despite being ancient, RPPs generally lack sequence conservation compared to other universal proteins. By analyzing the relative frequency of residues at every position in the context of the high-resolution structures of each of the RPPs (either alone or as functional binary complexes), we suggest residues for mutational analysis that may help uncover structure-function relationships in RPPs. PMID:27104580

  8. Knowledge of Native Protein-Protein Interfaces Is Sufficient To Construct Predictive Models for the Selection of Binding Candidates.

    PubMed

    Popov, Petr; Grudinin, Sergei

    2015-10-26

    Selection of putative binding poses is a challenging part of virtual screening for protein-protein interactions. Predictive models to filter out binding candidates with the highest binding affinities comprise scoring functions that assign a score to each binding pose. Existing scoring functions are typically deduced by collecting statistical information about interfaces of native conformations of protein complexes along with interfaces of a large generated set of non-native conformations. However, the obtained scoring functions become biased toward the method used to generate the non-native conformations, i.e., they may not recognize near-native interfaces generated with a different method. The present study demonstrates that knowledge of only native protein-protein interfaces is sufficient to construct well-discriminative predictive models for the selection of binding candidates. Here we introduce a new scoring method that comprises a knowledge-based potential called KSENIA deduced from structural information about the native interfaces of 844 crystallographic protein-protein complexes. We derive KSENIA using convex optimization with a training set composed of native protein complexes and their near-native conformations obtained using deformations along the low-frequency normal modes. As a result, our knowledge-based potential has only marginal bias toward a method used to generate putative binding poses. Furthermore, KSENIA is smooth by construction, which allows it to be used along with rigid-body optimization to refine the binding poses. Using several test benchmarks, we demonstrate that our method discriminates well native and near-native conformations of protein complexes from non-native ones. Our methodology can be easily adapted to the recognition of other types of molecular interactions, such as protein-ligand, protein-RNA, etc. KSENIA will be made publicly available as a part of the SAMSON software platform at https://team.inria.fr/nano-d/software .

  9. Comparison of positional surfactant isomers for displacement of rubisco protein from the air-water interface.

    PubMed

    He, Lizhong; Onaizi, Sagheer A; Dimitrijev-Dwyer, Mirjana; Malcolm, Andrew S; Shen, Hsin-Hui; Dong, Chuchuan; Holt, Stephen A; Thomas, Robert K; Middelberg, Anton P J

    2011-08-15

    Protein-surfactant interaction, which is a function of the protein and surfactant characteristics, is a common phenomenon in a wide range of industrial applications. In this work, we used rubisco, the most abundant protein in nature, as a model protein and sodium dodecylbenzenesulfonate (SDOBS), one of the most widely used commercial surfactants, with two positional isomers (SDOBS-2 and SDOBS-6), as a model surfactant. We first examined the surface tension and the mechanical properties of interfacial mixed rubisco-SDOBS films adsorbed at the air-water interface. The concentration of rubisco in solution was fixed at 0.1 mg mL(-1) while the SDOBS concentration varied from 0 to 150 μM. Both the surface tension and the mechanical strength of the interfacial film decreased with increasing SDOBS concentration. Overall, the surface tension of a rubisco-SDOBS-6 mixture is lower than that of rubisco-SDOBS-2, while the mechanical strength of both systems is similar. Neutron reflection data suggest that rubisco protein is likely denatured at the interface. The populations of rubisco and SDOBS of the mixed systems at the interface were determined by combining non-deuterated and deuterated SDOBS to provide contrast variation. At a low surfactant concentration, SDOBS-6 has a stronger ability to displace rubisco from the air-water interface than SDOBS-2. However, when surfactant concentration reaches 50 μM, SDOBS-2 has a higher population than SDOBS-6, with more rubisco displaced from the interface. The results presented in this work suggest that the extent of protein displacement from the air-water interface, and hence the nature of the protein-surfactant interactions at the interface, are strongly affected by the position of surfactant isomerisation, which might allow the design of formulations for efficient removal of protein stains.

  10. Cholera toxin B subunit-five-stranded α-helical coiled-coil fusion protein: "five-to-five" molecular chimera displays robust physicochemical stability.

    PubMed

    Arakawa, Takeshi; Harakuni, Tetsuya

    2014-09-03

    To create a physicochemically stable cholera toxin (CT) B subunit (CTB), it was fused to the five-stranded α-helical coiled-coil domain of cartilage oligomeric matrix protein (COMP). The chimeric fusion protein (CTB-COMP) was expressed in Pichia pastoris, predominantly as a pentamer, and retained its affinity for the monosialoganglioside GM1, a natural receptor of CT. The fusion protein displayed thermostability, tolerating the boiling temperature of water for 10min, whereas unfused CTB readily dissociated to its monomers and lost its affinity for GM1. The fusion protein also displayed resistance to strong acid at pHs as low as 0.1, and to the protein denaturant sodium dodecyl sulfate at concentrations up to 10%. Intranasal administration of the fusion protein to mice induced anti-B subunit serum IgG, even after the protein was boiled, whereas unfused CTB showed no thermostable mucosal immunogenicity. This study demonstrates that CTB fused to a pentameric α-helical coiled coil has a novel physicochemical phenotype, which may provide important insight into the molecular design of enterotoxin-B-subunit-based vaccines and vaccine delivery molecules.

  11. Chemomechanical Coupling in Hexameric Protein-Protein Interfaces Harnesses Energy within V-Type ATPases.

    PubMed

    Singharoy, Abhishek; Chipot, Christophe; Moradi, Mahmoud; Schulten, Klaus

    2017-01-11

    ATP synthase is the most prominent bioenergetic macromolecular motor in all life forms, utilizing the proton gradient across the cell membrane to fuel the synthesis of ATP. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, the precise molecular mechanism whereby vacuolar (V-type) ATP synthase fulfills its biological function remains largely fragmentary. Recently, crystallographers provided the first high-resolution view of ATP activity in Enterococcus hirae V1-ATPase. Employing a combination of transition-path sampling and high-performance free-energy methods, the sequence of conformational transitions involved in a functional cycle accompanying ATP hydrolysis has been investigated in unprecedented detail over an aggregate simulation time of 65 μs. Our simulated pathways reveal that the chemical energy produced by ATP hydrolysis is harnessed via the concerted motion of the protein-protein interfaces in the V1-ring, and is nearly entirely consumed in the rotation of the central stalk. Surprisingly, in an ATPase devoid of a central stalk, the interfaces of this ring are perfectly designed for inducing ATP hydrolysis. However, in a complete V1-ATPase, the mechanical property of the central stalk is a key determinant of the rate of ATP turnover. The simulations further unveil a sequence of events, whereby unbinding of the hydrolysis product (ADP + Pi) is followed by ATP uptake, which, in turn, leads to the torque generation step and rotation of the center stalk. Molecular trajectories also bring to light multiple intermediates, two of which have been isolated in independent crystallography experiments.

  12. Cavbeta-subunit displacement is a key step to induce the reluctant state of P/Q calcium channels by direct G protein regulation.

    PubMed

    Sandoz, Guillaume; Lopez-Gonzalez, Ignacio; Grunwald, Didier; Bichet, Delphine; Altafaj, Xavier; Weiss, Norbert; Ronjat, Michel; Dupuis, Alain; De Waard, Michel

    2004-04-20

    P/Q Ca(2+) channel activity is inhibited by G protein-coupled receptor activation. Channel inhibition requires a direct Gbetagamma binding onto the pore-forming subunit, Ca(v)2.1. It is characterized by biophysical changes, including current amplitude reduction, activation kinetic slowing, and an I-V curve shift, which leads to a reluctant mode. Here, we have characterized the contribution of the auxiliary beta(3)-subunit to channel regulation by G proteins. The shift in I-V to a P/Q reluctant mode is exclusively observed in the presence of beta(3). Along with the observation that Gbetagamma has no effect on the I-V curve of Ca(v)2.1 alone, we propose that the reluctant mode promoted by Gbetagamma corresponds to a state in which the beta(3)-subunit has been displaced from its channel-binding site. We validate this hypothesis with a beta(3)-I-II(2.1) loop chimera construct. Gbetagamma binding onto the I-II(2.1) loop portion of the chimera releases the beta(3)-binding domain and makes it available for binding onto the I-II loop of Ca(v)1.2, a G protein-insensitive channel. This finding is extended to the full-length Ca(v)2.1 channel by using fluorescence resonance energy transfer. Gbetagamma injection into Xenopus oocytes displaces a Cy3-labeled beta(3)-subunit from a GFP-tagged Ca(v)2.1 channel. We conclude that beta-subunit dissociation from the channel complex constitutes a key step in P/Q calcium channel regulation by G proteins that underlies the reluctant state and is an important process for modulating neurotransmission through G protein-coupled receptors.

  13. Small angle X-ray scattering of wheat seed-storage proteins: alpha-, gamma- and omega-gliadins and the high molecular weight (HMW) subunits of glutenin.

    PubMed

    Thomson, N H; Miles, M J; Popineau, Y; Harries, J; Shewry, P; Tatham, A S

    1999-03-19

    Small angle X-ray scattering in solution was performed on seed-storage proteins from wheat. Three different groups of gliadins (alpha-, gamma- and omega-) and a high molecular weight (HMW) subunit of glutenin (1Bx20) were studied to determine molecular size parameters. All the gliadins could be modelled as prolate ellipsoids with extended conformations. The HMW subunit existed as a highly extended rod-like particle in solution with a length of about 69 nm and a diameter of about 6.4 nm. Specific aggregation effects were observed which may reflect mechanisms of self-assembly that contribute to the unique viscoelastic properties of wheat dough.

  14. Interface interactions of natural rubber and protein/fiber aggregates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mechanical properties of natural rubber are improved with a renewable filler for rubber applications. Aggregates of protein and fiber that constitute soy protein concentrate were shear-reduced and used to enhance the tensile modulus of the natural rubber. The aqueous dispersion of the shear-reduced ...

  15. Weak conservation of structural features in the interfaces of homologous transient protein–protein complexes

    PubMed Central

    Sudha, Govindarajan; Singh, Prashant; Swapna, Lakshmipuram S; Srinivasan, Narayanaswamy

    2015-01-01

    Residue types at the interface of protein–protein complexes (PPCs) are known to be reasonably well conserved. However, we show, using a dataset of known 3-D structures of homologous transient PPCs, that the 3-D location of interfacial residues and their interaction patterns are only moderately and poorly conserved, respectively. Another surprising observation is that a residue at the interface that is conserved is not necessarily in the interface in the homolog. Such differences in homologous complexes are manifested by substitution of the residues that are spatially proximal to the conserved residue and structural differences at the interfaces as well as differences in spatial orientations of the interacting proteins. Conservation of interface location and the interaction pattern at the core of the interfaces is higher than at the periphery of the interface patch. Extents of variability of various structural features reported here for homologous transient PPCs are higher than the variation in homologous permanent homomers. Our findings suggest that straightforward extrapolation of interfacial nature and inter-residue interaction patterns from template to target could lead to serious errors in the modeled complex structure. Understanding the evolution of interfaces provides insights to improve comparative modeling of PPC structures. PMID:26311309

  16. Cloning and characterization of genes encoding alpha and beta subunits of glutamate-gated chloride channel protein in Cylicocyclus nassatus.

    PubMed

    Tandon, Ritesh; LePage, Keith T; Kaplan, Ray M

    2006-11-01

    The invertebrate glutamate-gated chloride channels (GluCls) are receptor molecules and targets for the avermectin-milbemycin (AM) group of anthelmintics. Mutations in GluCls are associated with ivermectin resistance in the soil dwelling nematode Caenorhabditis elegans and the parasitic nematode Cooperia oncophora. In this study, full-length cDNAs encoding alpha and beta subunits of GluCl were cloned and sequenced in Cylicocyclus nassatus, a common and important cyathostomin nematode parasite of horses. Both genes possess the sequence characteristics typical of GluCls, and phylogenetic analysis confirms that these genes are evolutionarily closely related to GluCls of other nematodes and flies. Complete coding sequences of C. nassatus GluCl-alpha and GluCl-beta were subcloned into pTL1 mammalian expression vector, and proteins were expressed in COS-7 cells. Ivermectin-binding characteristics were determined by incubating COS-7 cell membranes expressing C. nassatus GluCl-alpha and GluCl-beta proteins with [(3)H]ivermectin. In competitive binding experiments, fitting the data to a one site competition model, C. nassatus GluCl-alpha was found to bind [(3)H]ivermectin with a high amount of displaceable binding (IC(50)=208 pM). Compared to the mock-transfected COS-7 cells, the means of [(3)H]ivermectin binding were significantly different for C. nassatus GluCl-alpha and the Haemonchus contortus GluCl (HcGluCla) (p=0.018 and 0.023, respectively) but not for C. nassatus GluCl-beta (p=0.370). This is the first report of orthologs of GluCl genes and in vitro expression of an ivermectin-binding protein in a cyathostomin species. These data suggest the likelihood of a similar mechanism of action of AM drugs in these parasites, and suggest that mechanisms of resistance may also be similar.

  17. Expression of a bioactive fusion protein of Escherichia coli heat-labile toxin B subunit to a synapsin peptide.

    PubMed

    Julia Scerbo, M; Bibolini, Mario J; Barra, José L; Roth, German A; Monferran, Clara G

    2008-06-01

    The B subunit of Escherichia coli heat-labile toxin (LTB) may function as an efficient carrier molecule for the delivery of genetically coupled antigens across the mucosal barrier. We constructed vectors for the expression of LTB and LTBSC proteins. LTBSC is a fusion protein that comprises the amino acid sequence from the C-domain of rat synapsin fused to the C-terminal end of LTB. Both constructions have a coding sequence for a 6His-tag fused in-frame. LTBSC was expressed in E. coli as inclusion bodies. The inclusion bodies were isolated and purified by Ni2+-chelating affinity chromatography under denaturing condition. Purified LTBSC was diluted in several refolding buffers to gain a soluble and biologically active protein. Refolded LTBSC assembled as an active oligomer which binds to the GM1 receptor in an enzyme-linked immunosorbent assay (ELISA). Soluble LTB in the E. coli lysate was also purified by Ni2+-chelating affinity chromatography and the assembled pentamer was able to bind with high affinity to GM1 in vitro. LTBSC and LTB were fed to rats and the ability to induce antigen-specific tolerance was tested. LTBSC inhibited the specific delayed-type hypersensitivity (DTH) response and induced decreased antigen-specific in vivo and in vitro cell proliferation more efficiently than LTB. Thus, the novel hybrid molecule LTBSC when orally delivered was able to elicit a systemic immune response. These results suggest that LTBSC could be suitable for exploring further therapeutic treatment of autoimmune inflammatory diseases involving antigens from central nervous system.

  18. The Ca2+-binding sequence in bovine brain S100b protein beta-subunit. A spectroscopic study.

    PubMed Central

    Baudier, J; Cole, R D

    1989-01-01

    Conformational changes in the beta-subunit of the bovine brain Ca2+-binding protein S100b (S100-beta) accompanying Ca2+ binding were investigated by analysis of the spectroscopic properties of the single tyrosine residue (Tyr17 beta) and flow-dialysis binding experiments. S100-beta binds Ca2+ sequentially at two sites to change the conformation of the protein. The first Ca2+ ion binds to site II beta, a typical Ca2+-binding site in the C-terminal region, and it does not significantly perturb the proximal environment of Tyr17 beta. After the first site is occupied, another Ca2+ ion binds to the N-terminal Ca2+-binding site, I beta, and strengthens a hydrogen bond between Tyr17 beta and a neighbouring carboxylate acceptor group, which results in a large increase in the Tyr17 beta fluorescence spectrum half-width and a positive absorption and c.d. signal between 290 and 275 nm. Ca2+ binding to the S100b.Zn2+6 complex, studied by flow-dialysis and fluorescence measurements showed that, although Zn2+ ions increase the affinity of S100b protein for Ca2+, the Ca2+-binding sequence was not changed. Tb3+ (terbium ion) binding studies on the S100b.Zn2+6 complex proved that Tb3+ antagonizes only Ca2+ binding site II beta and confirmed the sequential occupation of Ca2+-binding sites on the S100b.Zn2+6 complex. PMID:2604719

  19. Defining A-Kinase Anchoring Protein (AKAP) Specificity for the Protein Kinase A Subunit RI (PKA-RI).

    PubMed

    Autenrieth, Karolin; Bendzunas, N George; Bertinetti, Daniela; Herberg, Friedrich W; Kennedy, Eileen J

    2016-04-15

    A-Kinase anchoring proteins (AKAPs) act as spatial and temporal regulators of protein kinase A (PKA) by localizing PKA along with multiple proteins into discrete signaling complexes. AKAPs interact with the PKA holoenzyme through an α-helix that docks into a groove formed on the dimerization/docking domain of PKA-R in an isoform-dependent fashion. In an effort to understand isoform selectivity at the molecular level, a library of protein-protein interaction (PPI) disruptors was designed to systematically probe the significance of an aromatic residue on the AKAP docking sequence for RI selectivity. The stapled peptide library was designed based on a high affinity, RI-selective disruptor of AKAP binding, RI-STAD-2. Phe, Trp and Leu were all found to maintain RI selectivity, whereas multiple intermediate-sized hydrophobic substitutions at this position either resulted in loss of isoform selectivity (Ile) or a reversal of selectivity (Val). As a limited number of RI-selective sequences are currently known, this study aids in our understanding of isoform selectivity and establishing parameters for discovering additional RI-selective AKAPs.

  20. Burial Level Change Defines a High Energetic Relevance for Protein Binding Interfaces.

    PubMed

    Li, Zhenhua; He, Ying; Wong, Limsoon; Li, Jinyan

    2015-01-01

    Protein-protein interfaces defined through atomic contact or solvent accessibility change are widely adopted in structural biology studies. But, these definitions cannot precisely capture energetically important regions at protein interfaces. The burial depth of an atom in a protein is related to the atom's energy. This work investigates how closely the change in burial level of an atom/residue upon complexation is related to the binding. Burial level change is different from burial level itself. An atom deeply buried in a monomer with a high burial level may not change its burial level after an interaction and it may have little burial level change. We hypothesize that an interface is a region of residues all undergoing burial level changes after interaction. By this definition, an interface can be decomposed into an onion-like structure according to the burial level change extent. We found that our defined interfaces cover energetically important residues more precisely, and that the binding free energy of an interface is distributed progressively from the outermost layer to the core. These observations are used to predict binding hot spots. Our approach's F-measure performance on a benchmark dataset of alanine mutagenesis residues is much superior or similar to those by complicated energy modeling or machine learning approaches.

  1. G-protein stimulatory subunit alpha and Gq/11α G-proteins are both required to maintain quiescent stem-like chondrocytes

    PubMed Central

    Chagin, Andrei S.; Vuppalapati, Karuna K.; Kobayashi, Tatsuya; Guo, Jun; Hirai, Takao; Chen, Min; Offermanns, Stefan; Weinstein, Lee S.; Kronenberg, Henry M.

    2014-01-01

    Round chondrocytes in the resting zone of the growth plate provide precursors for columnar chondrocytes and have stem-like properties. Here we demonstrate that these stem-like chondrocytes undergo apoptosis in the absence of the receptor (PPR) for parathyroid hormone-related protein. We examine the possible roles of heterotrimeric G-proteins activated by the PPR. Inactivation of the G-protein stimulatory α-subunit (Gsα) leads to accelerated differentiation of columnar chondrocytes, as seen in the PPR knockout, but a remnant of growth cartilage remains, in contrast to disappearance of the growth cartilage in the PPR knockout. Stem-like chondrocytes lose their quiescence and proliferate upon Gsα ablation. Inactivation of Gsα in mice with a mutant PPR that cannot activate G proteins, Gq and G11, leads to a PPR knockout-like phenotype. Thus, Gsα is the major mediator of the anti-differentiation action of the PPR, while activation of both Gsα and Gq/11α is required for quiescence of stem-like chondrocytes. PMID:24781502

  2. Engineering A-kinase anchoring protein (AKAP)-selective regulatory subunits of protein kinase A (PKA) through structure-based phage selection.

    PubMed

    Gold, Matthew G; Fowler, Douglas M; Means, Christopher K; Pawson, Catherine T; Stephany, Jason J; Langeberg, Lorene K; Fields, Stanley; Scott, John D

    2013-06-14

    PKA is retained within distinct subcellular environments by the association of its regulatory type II (RII) subunits with A-kinase anchoring proteins (AKAPs). Conventional reagents that universally disrupt PKA anchoring are patterned after a conserved AKAP motif. We introduce a phage selection procedure that exploits high-resolution structural information to engineer RII mutants that are selective for a particular AKAP. Selective RII (RSelect) sequences were obtained for eight AKAPs following competitive selection screening. Biochemical and cell-based experiments validated the efficacy of RSelect proteins for AKAP2 and AKAP18. These engineered proteins represent a new class of reagents that can be used to dissect the contributions of different AKAP-targeted pools of PKA. Molecular modeling and high-throughput sequencing analyses revealed the molecular basis of AKAP-selective interactions and shed new light on native RII-AKAP interactions. We propose that this structure-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces.

  3. Exploring protein interfaces with a general photochemical reagent.

    PubMed

    Gómez, Gabriela E; Cauerhff, Ana; Craig, Patricio O; Goldbaum, Fernando A; Delfino, José M

    2006-04-01

    Protein folding, natural conformational changes, or interaction between partners involved in recognition phenomena brings about differences in the solvent-accessible surface area (SASA) of the polypeptide chain. This primary event can be monitored by the differential chemical reactivity of functional groups along the protein sequence. Diazirine (DZN), a photoreactive gas similar in size to water, generates methylene carbene (:CH(2)). The extreme chemical reactivity of this species allows the almost instantaneous and indiscriminate modification of its immediate molecular cage. (3)H-DZN was successfully used in our laboratory for studying protein structure and folding. Here we address for the first time the usefulness of this probe to examine the area of interaction in protein-protein complexes. For this purpose we chose the complex formed between hen egg white lysozyme (HEWL) and the monoclonal antibody IgG(1) D1.3. :CH(2) labeling of free HEWL or complexed with IgG(1) D1.3 yields 2.76 and 2.32 mmol CH(2) per mole protein at 1 mM DZN concentration, respectively. This reduction (15%) becomes consistent with the expected decrement in the SASA of HEWL occurring upon complexation derived from crystallographic data (11%), in agreement with the known unspecific surface labeling reaction of :CH(2). Further comparative analysis at the level of tryptic peptides led to the identification of the sites involved in the interaction. Remarkably, those peptides implicated in the contact area show the highest differential labeling: H(15)GLDNYR(21), G(117)TDVQAWIR(125), andG(22)YSLGNWVCAAK(33). Thus, protein footprinting with DZN emerges as a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.

  4. Molecular characterisation of cAMP-dependent protein kinase (PK-A) catalytic subunit isoforms in the male tick, Amblyomma hebraeum.

    PubMed

    Tabish, Mohammad; Clegg, Roger A; Turner, Philip C; Jonczy, Jan; Rees, Huw H; Fisher, Michael J

    2006-12-01

    The cAMP-dependent protein kinase (protein kinase A, PK-A) plays a central role in the regulation of diverse aspects of cellular activity. Specifically, PK-A appears to play a key controlling role in the maturation of spermatids. Using a PCR-based approach, with degenerate primers from the highly conserved regions of the PK-A catalytic (C) subunit in combination with 5' and 3' RACE, we have cloned three cDNAs for the PK-A C-subunit of the male tick, Amblyomma hebraeum. The three cDNAs have open reading frames of 1059, 1275 and 1404bp which encode proteins of 40.6, 48.2 and 52.5kDa, respectively. These transcripts appear to arise from 5' alternative splicing of RNA derived from a single gene for the PK-A C-subunit. One isoform (AH-PK-A C1), in common with PK-A C-subunits from a range of species, contains a consensus sequence for N-myristoylation. RT-PCR and Western blot experiments suggest that the three splice variants are expressed ubiquitously; however, expression of the myristoylatable AH-PK-A C1 isoform is predominant in all investigated tissues (accessory gland, midgut, Malpighian tubules, salivary gland, testis and immature spermatids). There is no evidence for a sperm-specific PK-A C-subunit (Cs) in tick sperm; however, tyrosine protein phosphorylation, previously shown to be modulated by PK-A activity during mammalian sperm maturation, was observed in tick sperm.

  5. Molecular cloning of a pancreatic islet-specific glucose-6-phosphatase catalytic subunit-related protein.

    PubMed

    Arden, S D; Zahn, T; Steegers, S; Webb, S; Bergman, B; O'Brien, R M; Hutton, J C

    1999-03-01

    A pancreatic islet-specific glucose-6-phosphatase-related protein (IGRP) was cloned using a subtractive cDNA expression cloning procedure from mouse insulinoma tissue. Two alternatively spliced variants that differed by the presence or absence of a 118-bp exon (exon IV) were detected in normal balb/c mice, diabetic ob/ob mice, and insulinoma tissue. The longer, 1901-bp full-length cDNA encoded a 355-amino acid protein (molecular weight 40,684) structurally related (50% overall identity) to the liver glucose-6-phosphatase and exhibited similar predicted transmembrane topology, conservation of catalytically important residues, and the presence of an endoplasmic reticulum retention signal. The shorter transcript encoded two possible open reading frames (ORFs), neither of which possessed His174, a residue thought to be the phosphoryl acceptor (Pan CJ, Lei KJ, Annabi B, Hemrika W, Chou JY: Transmembrane topology of glucose-6-phosphatase. J Biol Chem 273:6144-6148, 1998). Northern blot and reverse transcription-polymerase chain reaction analysis showed that the mRNA was highly expressed in pancreatic islets and expressed more in beta-cell lines than in an alpha-cell line. It was notably absent in tissues and cell lines of non-islet neuroendocrine origin, and no other major tissue source of the mRNA was found. During development, it was expressed in parallel with insulin mRNA. The mRNA was efficiently translated and glycosylated in an in vitro translation/membrane translocation system and readily transcribed into COS 1, HIT, and CHO cells using cytomegalovirus or Rous sarcoma virus promoters. Whereas the liver glucose-6-phosphatase showed activity in these transfection systems, the IGRP failed to show glucose phosphotransferase or phosphatase activity with p-nitrophenol phosphate, inorganic pyrophosphate, or a range of sugar phosphates hydrolyzed by the liver enzyme. While the metabolic function of the enzyme is not resolved, its remarkable tissue-specific expression

  6. In vitro selection and prediction of TIP47 protein-interaction interfaces.

    PubMed

    Burguete, Alondra Schweizer; Harbury, Pehr B; Pfeffer, Suzanne R

    2004-10-01

    We present a new method for the rapid identification of amino acid residues that contribute to protein-protein interfaces. Tail-interacting protein of 47 kDa (TIP47) binds Rab9 GTPase and the cytoplasmic domains of mannose 6-phosphate receptors and is required for their transport from endosomes to the Golgi apparatus. Cysteine mutations were incorporated randomly into TIP47 by expression in Escherichia coli cells harboring specific misincorporator tRNAs. We made use of the ability of the native TIP47 protein to protect 48 cysteine probes from chemical modification by iodoacetamide as a means to obtain a surface map of TIP47, revealing the identity of surface-localized, hydrophobic residues that are likely to participate in protein-protein interactions. Direct mutation of predicted interface residues confirmed that the protein had altered binding affinity for the mannose 6-phosphate receptor. TIP47 mutants with enhanced or diminished affinities were also selected by affinity chromatography. These methods were validated in comparison with the protein's crystal structure, and provide a powerful means to predict protein-protein interaction interfaces.

  7. Chiral Vibrational Structures of Proteins at Interfaces Probed by Sum Frequency Generation Spectroscopy

    PubMed Central

    Fu, Li; Wang, Zhuguang; Yan, Elsa C.Y.

    2011-01-01

    We review the recent development of chiral sum frequency generation (SFG) spectroscopy and its applications to study chiral vibrational structures at interfaces. This review summarizes observations of chiral SFG signals from various molecular systems and describes the molecular origins of chiral SFG response. It focuses on the chiral vibrational structures of proteins and presents the chiral SFG spectra of proteins at interfaces in the C-H stretch, amide I, and N-H stretch regions. In particular, a combination of chiral amide I and N-H stretches of the peptide backbone provides highly characteristic vibrational signatures, unique to various secondary structures, which demonstrate the capacity of chiral SFG spectroscopy to distinguish protein secondary structures at interfaces. On the basis of these recent developments, we further discuss the advantages of chiral SFG spectroscopy and its potential application in various fields of science and technology. We conclude that chiral SFG spectroscopy can be a new approach to probe chiral vibrational structures of protein at interfaces, providing structural and dynamic information to study in situ and in real time protein structures and dynamics at interfaces. PMID:22272140

  8. Measuring interactions between polydimethylsiloxane and serum proteins at the air-water interface.

    PubMed

    Liao, Zhengzheng; Hsieh, Wan-Ting; Baumgart, Tobias; Dmochowski, Ivan J

    2013-07-30

    The interaction between synthetic polymers and proteins at interfaces is relevant to basic science as well as a wide range of applications in biotechnology and medicine. One particularly common and important interface is the air-water interface (AWI). Due to the special energetics and dynamics of molecules at the AWI, the interplay between synthetic polymer and protein can be very different from that in bulk solution. In this paper, we applied the Langmuir-Blodgett technique and fluorescence microscopy to investigate how the compression state of polydimethylsiloxane (PDMS) film at the AWI affects the subsequent adsorption of serum protein [e.g., human serum albumin (HSA) or immunoglobulin G (IgG)] and the interaction between PDMS and protein. Of particular note is our observation of circular PDMS domains with micrometer diameters that form at the AWI in the highly compressed state of the surface film: proteins were shown to adsorb preferentially to the surface of these circular PDMS domains, accompanied by a greater than 4-fold increase in protein found in the interfacial film. The PDMS-only film and the PDMS-IgG composite film were transferred to cover glass, and platinum-carbon replicas of the transferred films were further characterized by scanning electron microscopy and atomic force microscopy. We conclude that the structure of the PDMS film greatly affects the amount and distribution of protein at the interface.

  9. [Isolation and study of the properties of the regulator subunit of cAMP-dependent protein kinase].

    PubMed

    Iurkiv, V A; Severin, E S; Petukhov, S P; Bulargina, T V

    1982-12-01

    The regulatory subunit of type II cAMP-dependent proteinkinase was isolated from cytosol of the rabbit small intestinal mucosa by affinity chromatography. The preparation contained 3 proteolytic enzymes and occurred in two forms differing as regards cAMP affinity. The cAMP-binding capacity of the preparation was equal to 17 nmol cAMP/mg protein. To study the topography of the cAMP-binding center, use was made of cAMP analogs. It was demonstrated that introduction of the substituents into the 8th position of the purine ring and substitution with respect to the N6-exoaminogroup affected insignificantly the analog affinity for the cAMP-binding center. At the same time the substituents introduced into the first position of the adenine base, into the area of the 2'-hydroxyl group of ribose and into the cyclophosphate part of the cAMP molecule considerably decreased the analog affinity for the regulatory center of type II cAMP-dependent proteinkinase.

  10. The Adaptor Protein-1 μ1B Subunit Expands the Repertoire of Basolateral Sorting Signal Recognition in Epithelial Cells

    PubMed Central

    Guo, Xiaoli; Mattera, Rafael; Ren, Xuefeng; Chen, Yu; Retamal, Claudio; González, Alfonso; Bonifacino, Juan S.

    2014-01-01

    SUMMARY An outstanding question in protein sorting is why polarized epithelial cells express two isoforms of the μ1 subunit of the AP-1 clathrin adaptor complex: the ubiquitous μ1A and the epithelial-specific μ1B. Previous studies led to the notion that μ1A and μ1B mediate basolateral sorting predominantly from the trans-Golgi network (TGN) and recycling endosomes, respectively. Using improved analytical tools, however, we find that μ1A and μ1B largely colocalize with each other. They also colocalize to similar extents with TGN and recycling endosome markers, as well as with basolateral cargoes transiting biosynthetic and endocytic-recycling routes. Instead, the two isoforms differ in their signal-recognition specificity. In particular, μ1B preferentially binds a subset of signals from cargoes that are sorted basolaterally in a μ1B-dependent manner. We conclude that expression of distinct μ1 isoforms in epithelial cells expands the repertoire of signals recognized by AP-1 for sorting of a broader range of cargoes to the basolateral surface. PMID:24229647

  11. Tabulation as a high-resolution alternative to coarse-graining protein interactions: Initial application to virus capsid subunits

    NASA Astrophysics Data System (ADS)

    Spiriti, Justin; Zuckerman, Daniel M.

    2015-12-01

    Traditional coarse-graining based on a reduced number of interaction sites often entails a significant sacrifice of chemical accuracy. As an alternative, we present a method for simulating large systems composed of interacting macromolecules using an energy tabulation strategy previously devised for small rigid molecules or molecular fragments [S. Lettieri and D. M. Zuckerman, J. Comput. Chem. 33, 268-275 (2012); J. Spiriti and D. M. Zuckerman, J. Chem. Theory Comput. 10, 5161-5177 (2014)]. We treat proteins as rigid and construct distance and orientation-dependent tables of the interaction energy between them. Arbitrarily detailed interactions may be incorporated into the tables, but as a proof-of-principle, we tabulate a simple α-carbon Gō-like model for interactions between dimeric subunits of the hepatitis B viral capsid. This model is significantly more structurally realistic than previous models used in capsid assembly studies. We are able to increase the speed of Monte Carlo simulations by a factor of up to 6700 compared to simulations without tables, with only minimal further loss in accuracy. To obtain further enhancement of sampling, we combine tabulation with the weighted ensemble (WE) method, in which multiple parallel simulations are occasionally replicated or pruned in order to sample targeted regions of a reaction coordinate space. In the initial study reported here, WE is able to yield pathways of the final ˜25% of the assembly process.

  12. NUCLEAR FACTOR Y, Subunit A (NF-YA) Proteins Positively Regulate Flowering and Act Through FLOWERING LOCUS T

    PubMed Central

    Siriwardana, Chamindika L.; Kumimoto, Roderick W.; Mantovani, Roberto

    2016-01-01

    Photoperiod dependent flowering is one of several mechanisms used by plants to initiate the developmental transition from vegetative growth to reproductive growth. The NUCLEAR FACTOR Y (NF-Y) transcription factors are heterotrimeric complexes composed of NF-YA and histone-fold domain (HFD) containing NF-YB/NF-YC, that initiate photoperiod-dependent flowering by cooperatively interacting with CONSTANS (CO) to drive the expression of FLOWERING LOCUS T (FT). This involves NF-Y and CO binding at distal CCAAT and proximal “CORE” elements, respectively, in the FT promoter. While this is well established for the HFD subunits, there remains some question over the potential role of NF-YA as either positive or negative regulators of this process. Here we provide strong support, in the form of genetic and biochemical analyses, that NF-YA, in complex with NF-YB/NF-YC proteins, can directly bind the distal CCAAT box in the FT promoter and are positive regulators of flowering in an FT-dependent manner. PMID:27977687

  13. Investigation of the G protein subunit Gαolf gene (GNAL) in attention deficit/hyperactivity disorder

    PubMed Central

    Laurin, Nancy; Ickowicz, Abel; Pathare, Tejaswee; Malone, Molly; Tannock, Rosemary; Schachar, Russell; Kennedy, James L.; Barr, Cathy L.

    2016-01-01

    The dopamine system plays an important role in the regulation of attention and motor behavior, subsequently, several dopamine-related genes have been associated with Attention Deficit/Hyperactivity Disorder (ADHD). Among them are the dopamine receptors D1 and D5 that mediate adenylyl cyclase activation through coupling with Gs-like proteins. We thus hypothesized that the Gs-like subunit Gαolf, expressed in D1-rich areas of the brain, contributes to the genetic susceptibility of ADHD. To evaluate the involvement of the Gαolf gene, GNAL, in ADHD, we examined the inheritance pattern of 12 GNAL polymorphisms in 258 nuclear families ascertained through a proband with ADHD (311 affected children) using the transmission/disequilibrium test (TDT). Categorical analysis of individual marker alleles demonstrated biased transmission of one polymorphism in GNAL intron 3 (rs2161961; P = 0.011). We also observed significant relationships between rs2161961 and dimensional symptoms of inattention and hyperactivity/impulsivity (P = 0.003 and P = 0.008). In addition, because of recent evidence of imprinting at the GNAL locus, secondary analyses were split into maternal and paternal transmissions to assess a contribution of parental effects. We found evidence of strong maternal effect, with preferential transmission of maternal alleles for rs2161961A (P = 0.005) and rs8098539A (P = 0.035). These preliminary findings suggest a possible contribution of GNAL in the susceptibility to ADHD, with possible involvement of parent-of-origin effects. PMID:17166517

  14. Identification of residues crucial for the interaction between human neuroglobin and the α-subunit of heterotrimeric Gi protein

    PubMed Central

    Takahashi, Nozomu; Wakasugi, Keisuke

    2016-01-01

    Mammalian neuroglobin (Ngb) protects neuronal cells under conditions of oxidative stress. We previously showed that human Ngb acts as a guanine nucleotide dissociation inhibitor (GDI) for the α-subunits of heterotrimeric Gi/o proteins and inhibits the decrease in cAMP concentration, leading to protection against cell death. In the present study, we used an eukaryotic expression vector driving high-level expression of human wild-type Ngb or Ngb mutants that either exhibit or lack GDI activities in human cells. We demonstrate that the GDI activity of human Ngb is tightly correlated with its neuroprotective activity. We further demonstrate that Glu53, Glu60, and Glu118 of human Ngb are crucial for both the neuroprotective activity and interaction with Gαi1. Moreover, we show that Lys46, Lys70, Arg208, Lys209, and Lys210 residues of Gαi1 are important for binding to human Ngb. We propose a molecular docking model of the complex between human Ngb and Gαi1. PMID:27109834

  15. Dehydroepiandrosterone promotes pulmonary artery relaxation by NADPH oxidation-elicited subunit dimerization of protein kinase G 1α

    PubMed Central

    Patel, Dhara; Kandhi, Sharath; Kelly, Melissa; Neo, Boon Hwa

    2013-01-01

    The activity of glucose-6-phosphate dehydrogenase (G6PD) controls a vascular smooth muscle relaxing mechanism promoted by the oxidation of cytosolic NADPH, which has been associated with activation of the 1α form of protein kinase G (PKG-1α) by a thiol oxidation-elicited subunit dimerization. This PKG-1α-activation mechanism appears to contribute to responses of isolated endothelium-removed bovine pulmonary arteries (BPA) elicited by peroxide, cytosolic NADPH oxidation resulting from G6PD inhibition, and hypoxia. Dehydroepiandrosterone (DHEA) is a steroid hormone with pulmonary vasodilator activity, which has beneficial effects in treating pulmonary hypertension. Because multiple mechanisms have been suggested for the vascular effects of DHEA and one of the known actions of DHEA is inhibiting G6PD, we investigated whether it promoted relaxation associated with NADPH oxidation, PKG-1α dimerization, and PKG activation detected by increased vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Relaxation of BPA to DHEA under aerobic or hypoxic conditions was associated with NADPH oxidation, PKG-1α dimerization, and increased VASP phosphorylation. The vasodilator activity of DHEA was markedly attenuated in pulmonary arteries and aorta from a PKG knockin mouse containing a serine in place of a cysteine involved in PKG dimerization. DHEA promoted increased PKG dimerization in lungs from wild-type mice, which was not detected in the PKG knockin mouse model. Thus PKG-1α dimerization is a major contributing factor to the vasodilator actions of DHEA and perhaps its beneficial effects in treating pulmonary hypertension. PMID:24375799

  16. The heterotrimeric G-protein β subunit, AGB1, plays multiple roles in the Arabidopsis salinity response.

    PubMed

    Yu, Yunqing; Assmann, Sarah M

    2015-10-01

    Salinity stress includes both osmotic and ionic toxicity. Sodium homeostasis is influenced by Na(+) uptake and extrusion, vacuolar Na(+) compartmentation and root to shoot Na(+) translocation via transpiration. The knockout mutant of the Arabidopsis heterotrimeric G-proteinsubunit, agb1, is hypersensitive to salt, exhibiting a leaf bleaching phenotype. We show that AGB1 is mainly involved in the ionic toxicity component of salinity stress and plays roles in multiple processes of Na(+) homeostasis. agb1 mutants accumulate more Na(+) and less K(+) in both shoots and roots of hydroponically grown plants, as measured by inductively coupled plasma atomic emission spectrometry. agb1 plants have higher root to shoot translocation rates of radiolabelled (24) Na(+) under transpiring conditions, as a result of larger stomatal apertures and increased stomatal conductance. (24) Na(+) tracer experiments also show that (24) Na(+) uptake rates by excised roots of agb1 and wild type are initially equal, but that agb1 has higher net Na(+) uptake at 90 min, implicating possible involvement of AGB1 in the regulation of Na(+) efflux. Calcium alleviates the salt hypersensitivity of agb1 by reducing Na(+) accumulation to below the toxicity threshold. Our results provide new insights into the regulatory pathways underlying plant responses to salinity stress, an important agricultural problem.

  17. A kinase anchoring protein (AKAP) interaction and dimerization of the RIalpha and RIbeta regulatory subunits of protein kinase a in vivo by the yeast two hybrid system.

    PubMed

    Carlson, Cathrine R; Ruppelt, Anja; Taskén, Kjetil

    2003-03-28

    Protein kinase A (PKA) regulatory (R) subunits dimerize through an N-terminal motif. Such dimerization is necessary for binding to PKA anchoring proteins (AKAPs) and targeting of PKA to its site of action. In the present study, we used the yeast two-hybrid system as an in vivo bio-reporter assay and analyzed the formation of homo- and heterodimeric complexes of RIalpha and RIbeta as well as AKAP binding of RI dimers. Native polyacrylamide gel electrophoresis (PAGE) of yeast extracts confirmed the two-hybrid data. Both RIalpha- and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer were observed. Single, double and one triple mutation were introduced into the RIalpha and RIbeta subunits and dimerization properties of the mutants were analyzed. Consistent with previous reports, RIalpha(C37H) dimerized, although the disulfide bridges were disrupted, whereas the additional mutation of F47 or F52 abolished the dimerization. Corresponding mutations (C38H, F48A, F53A) in RIbeta were not sufficient to abolish the RIbeta dimerization, indicating that additional or other amino acids are important. RIalpha:RIbeta heterodimers of the mutants were formed at intermediate stringency. Analysis of ternary complexes by the yeast two-hybrid system revealed that RIalpha and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer and several of the mutants were able to bind to the R-binding domain of AKAP149/D-AKAP1. Furthermore, an RIbeta:AKAP149 complex was identified following introduction of RIbeta into HEK293 cells. Importantly, RIbeta revealed AKAP binding properties similar to those of RIalpha, indicating that RIbeta holoenzymes may be anchored.

  18. Determination of disulfide array and subunit structure of taste-modifying protein, miraculin.

    PubMed

    Igeta, H; Tamura, Y; Nakaya, K; Nakamura, Y; Kurihara, Y

    1991-09-20

    The taste-modifying protein, miraculin (Theerasilp, S. et al. (1989) J. Biol. Chem. 264, 6655-6659) has seven cysteine residues in a molecule composed of 191 amino acid residues. The formation of three intrachain disulfide bridges at Cys-47-Cys-92, Cys-148-Cys-159 and Cys-152-Cys-155 and one interchain disulfide bridge at Cys-138 was determined by amino acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. The presence of an interchain disulfide bridge was also supported by the fact that the cystine peptide containing Cys-138 showed a negative color test for the free sulfhydryl group and a positive test after reduction with dithiothreitol. The molecular mass of non-reduced miraculin (43 kDa) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was nearly twice the calculated molecular mass based on the amino acid sequence and the carbohydrate content of reduced miraculin (25 kDa). The molecular mass of native miraculin determined by low-angle laser light scattering was 90 kDa. Application of a crude extract of miraculin to a Sephadex G-75 column indicated that the taste-modifying activity appears at 52 kDa. It was concluded that native miraculin in pure form is a tetramer of the 25 kDa-peptide and native miraculin in crude state or denatured, non-reduced miraculin in pure form is a dimer of the peptide. Both tetramer miraculin and native dimer miraculin in crude state had the taste-modifying activity.

  19. Viscoelastic Characterization of Gels at Metal-Protein Interfaces

    NASA Astrophysics Data System (ADS)

    Martin, Elizabeth; Shull, Kenneth

    2015-03-01

    The interfacial gelation of proteins at metallic surfaces was investigated with an electrochemical quartz crystal microbalance (QCM). When Cr electrodes were corroded in proteinaceous solutions, it was found that gels will form at the Cr surfaces if molybdate ions are also present in the solution. A similar film will form on Cr when the proteins are replaced with a poly(allylamine) polyelectrolyte, suggesting that the gelation is due to a cross-linking reaction between the protein amine groups and the molybdate ions. Further, a method was developed to characterize the viscoelastic properties of thin polymeric films in liquid media using the QCM as a high frequency rheometer. By measuring the frequency and dissipation at multiple harmonics of the resonant frequency, the viscoelastic phase angle, density --modulus product, and mass per unit area of a film can be determined. The method was applied to characterize the protein films, demonstrating that they have a phase angle near 80° and a density --modulus product of ~107 Pa-g/cm3. Data imply that the gels are comprised of a weak proteinaceous network and exhibit similar mechanical properties as solutions containing 50 wt% protein. This project was funded by NSF Grant CMMI-1200529.

  20. Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases

    PubMed Central

    Park, Hyun-Joo; Tailor, Chetankumar; Che, Clare; Kamani, Mustafa; Spitalny, George; Binnington, Beth

    2016-01-01

    Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subunit has evolved to mimic a misfolded protein and hijack the ERAD membrane translocon (dislocon), to effect cytosolic access and cytopathology. We show such toxins compete for ERAD to rescue endogenous misfolded proteins. Cholera toxin or verotoxin (Shiga toxin) containing genetically inactivated (± an N-terminal polyleucine tail) A subunit can, within 2–4 hrs, temporarily increase F508delCFTR protein, the major cystic fibrosis (CF) mutant (5-10x), F508delCFTR Golgi maturation (<10x), cell surface expression (20x) and chloride transport (2x) in F508del CFTR transfected cells and patient-derived F508delCFTR bronchiolar epithelia, without apparent cytopathology. These toxoids also increase glucocerobrosidase (GCC) in N370SGCC Gaucher Disease fibroblasts (3x), another ERAD–exacerbated misfiling disease. We identify a new, potentially benign approach to the treatment of certain genetic protein misfolding diseases. PMID:27935997

  1. How good are state-of-the-art docking tools in predicting ligand binding modes in protein-protein interfaces?

    PubMed

    Krüger, Dennis M; Jessen, Gisela; Gohlke, Holger

    2012-11-26

    Protein-protein interfaces (PPIs) are an important class of drug targets. We report on the first large-scale validation study on docking into PPIs. DrugScore-adapted AutoDock3 and Glide showed good success rates with a moderate drop-off compared to docking to "classical targets". An analysis of the binding energetics in a PPI allows identifying those interfaces that are amenable for docking. The results are important for deciding if structure-based design approaches can be applied to a particular PPI.

  2. A multi-subunit based, thermodynamically stable model vaccine using combined immunoinformatics and protein structure based approach.

    PubMed

    Rana, Aarti; Akhter, Yusuf

    2016-04-01

    Immunizations with the conventional vaccines have failed to effectively inhibit the incidences and further dissemination of the infections. To address it, we have implemented protein structure based strategies to design an efficient multi-epitope subunit vaccine against Mycobacterium avium subsp. paratuberculosis (MAP). Previously reported immunodominant peptide epitope sequences from MAP1611 protein were conjugated together with a stretch of conserved amino acid residues of heparin-binding hemagglutinin, reported as a TLR4 agonist and was employed as an adjuvant to polarize the cellular responses toward host protective Th1 responses. These three types of component peptides were combined with the help of relevant linkers for efficient separation to improve and intensify the antigen processing and presentation. The primary structures of these multi peptides were 3-dimensional homology modeled to yield the final chimeric vaccine. Further, its conformational correctness and stability enhancement was assessed using molecular dynamics (MD) simulations. Finally, disulfide engineering in the most flexible regions of the molecule yielded three potential mutants, Y593C-E610C, Q631C-A634C and a double mutant Q631C-A634C/Y593C-E610C. The double mutant represents thermodynamically most stable version among them. It is potentially highly antigenic, soluble and non-allergen molecule interacting with the TLR receptor expressed on the immune cells. This vaccine contains both T-cell and several B-cell epitopes and an adjuvant which potentially possess protective cellular and humoral immune responses triggering properties. The presented vaccine strategy will be proven a promising pathogen specific candidate with wide therapeutic application against MAP which may be extended to other prevalent infections in future.

  3. G-Protein βγ Subunit Dimers Modulate Kidney Repair after Ischemia-Reperfusion Injury in Rats

    PubMed Central

    White, Sarah M.; North, Lauren M.; Haines, Emily; Goldberg, Megan; Sullivan, Lydia M.; Pressly, Jeffrey D.; Weber, David S.

    2014-01-01

    Heterotrimeric G-proteins play a crucial role in the control of renal epithelial cell function during homeostasis and in response to injury. In this report, G-protein βγ subunit (Gβγ) dimer activity was evaluated during the process of tubular repair after renal ischemia-reperfusion injury (IRI) in male Sprague Dawley rats. Rats were treated with a small molecule inhibitor of Gβγ activity, gallein (30 or 100 mg/kg), 1 hour after reperfusion and every 24 hours for 3 additional days. After IRI, renal dysfunction was prolonged after the high-dose gallein treatment in comparison with vehicle treatment during the 7-day recovery period. Renal tubular repair in the outer medulla 7 days after IRI was significantly (P < 0.001) attenuated after treatment with high-dose gallein (100 mg/kg) in comparison with low-dose gallein (30 mg/kg), or the vehicle and fluorescein control groups. Gallein treatment significantly reduced (P < 0.05) the number of proliferating cell nuclear antigen–positive tubular epithelial cells at 24 hours after the ischemia-reperfusion phase in vivo. In vitro application of gallein on normal rat kidney (NRK-52E) proximal tubule cells significantly reduced (P < 0.05) S-phase cell cycle entry compared with vehicle-treated cells as determined by 5′-bromo-2′-deoxyuridine incorporation. Taken together, these data suggest that Gβγ signaling contributes to the maintenance and repair of renal tubular epithelium and may be a novel therapeutic target for the development of drugs to treat acute kidney injury. PMID:25028481

  4. A study of the interface strength between protein and mineral in biological materials.

    PubMed

    Ji, Baohua

    2008-01-01

    Bone, tooth, mineralized tendon and sea shells are nanocomposites of protein and mineral with superior mechanical properties. As the mineral is so small at nanoscale, the volume fraction of the protein-mineral interface in the bulk materials can be enormously large; therefore, the mechanics of the interface should be critically important for the integrity of these biomaterials. Currently, people do not have a good understanding of the interface between protein and mineral, a hybrid interface between organic and inorganic constituents in biological materials. In this paper, a tension-shear chain (TSC) model is introduced into the Dugdale model for estimating the fracture energy of biomaterials. The strength of the hybrid interface is then studied with a "soft-hard" bi-layer fracture model, by which we find for the first time that the interface strength depends on both the size and geometry of the mineral crystal, and has been highly optimized through the miniaturization of mineral at nanoscale. This study may provide important insights into the mechanics of bone and tooth at small scale for tissue engineering in biomedical applications.

  5. Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons.

    PubMed

    Johar, Kaid; Priya, Anusha; Dhar, Shilpa; Liu, Qiuli; Wong-Riley, Margaret T T

    2013-11-01

    Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons.

  6. Protein Solvation in Membranes and at Water-Membrane Interfaces

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Chipot, Christophe; Wilson, Michael A.

    2002-01-01

    Different salvation properties of water and membranes mediate a host of biologically important processes, such as folding, insertion into a lipid bilayer, associations and functions of membrane proteins. These processes will be discussed in several examples involving synthetic and natural peptides. In particular, a mechanism by which a helical peptide becomes inserted into a model membrane will be described. Further, the molecular mechanism of recognition and association of protein helical segments in membranes will be discussed. These processes are crucial for proper functioning of a cell. A membrane-spanning domain of glycophorin A, which exists as a helical dimer, serves as the model system. For this system, the free energy of dissociation of the helices is being determined for both the wild type and a mutant, in which dimerization is disrupted.

  7. Topological transformations in proteins: effects of heating and proximity of an interface

    PubMed Central

    Zhao, Yani; Chwastyk, Mateusz; Cieplak, Marek

    2017-01-01

    Using a structure-based coarse-grained model of proteins, we study the mechanism of unfolding of knotted proteins through heating. We find that the dominant mechanisms of unfolding depend on the temperature applied and are generally distinct from those identified for folding at its optimal temperature. In particular, for shallowly knotted proteins, folding usually involves formation of two loops whereas unfolding through high-temperature heating is dominated by untying of single loops. Untying the knots is found to generally precede unfolding unless the protein is deeply knotted and the heating temperature exceeds a threshold value. We then use a phenomenological model of the air-water interface to show that such an interface can untie shallow knots, but it can also make knots in proteins that are natively unknotted. PMID:28051124

  8. Evolution of the Immune Response against Recombinant Proteins (TcpA, TcpB, and FlaA) as a Candidate Subunit Cholera Vaccine.

    PubMed

    Molaee, Neda; Mosayebi, Ghasem; Amozande-Nobaveh, Alireza; Soleyman, Mohammad Reza; Abtahi, Hamid

    2017-01-01

    Vibrio cholerae is the causative agent of cholera and annually leads to death of thousands of people around the globe. Two factors in the pathogenesis of this bacterium are its pili and flagella. The main subunits of pili TcpA, TcpB, and FlaA are the constituent subunit of flagella. In this study, we studied the ability of pili and flagella subunits to stimulate immune responses in mice. After amplification of TcpA, TcpB, and FlaA genes using PCR, they were cloned in expression plasmids. After production of the above-mentioned proteins by using IPTG, the proteins were purified and then approved using immunoblot method. After injection of the purified proteins to a mice model, immune response stimulation was evaluated by measuring the levels of IgG1 and IgG2a antibody titers, IL5 and IFN-γ. Immune response stimulation against pili and flagella antigens was adequate. Given the high levels of IL5 titer and IgG1 antibody, the stimulated immune response was toward Th1. Humoral immune response stimulation is of key importance in prevention of cholera. Our immunological analysis shows the appropriate immune response in mice model after vaccination with recombinant proteins. The high level of IL5 and low level of IFN-γ show the activation of Th2 cell response.

  9. Evolution of the Immune Response against Recombinant Proteins (TcpA, TcpB, and FlaA) as a Candidate Subunit Cholera Vaccine

    PubMed Central

    Molaee, Neda; Amozande-Nobaveh, Alireza; Soleyman, Mohammad Reza

    2017-01-01

    Vibrio cholerae is the causative agent of cholera and annually leads to death of thousands of people around the globe. Two factors in the pathogenesis of this bacterium are its pili and flagella. The main subunits of pili TcpA, TcpB, and FlaA are the constituent subunit of flagella. In this study, we studied the ability of pili and flagella subunits to stimulate immune responses in mice. After amplification of TcpA, TcpB, and FlaA genes using PCR, they were cloned in expression plasmids. After production of the above-mentioned proteins by using IPTG, the proteins were purified and then approved using immunoblot method. After injection of the purified proteins to a mice model, immune response stimulation was evaluated by measuring the levels of IgG1 and IgG2a antibody titers, IL5 and IFN-γ. Immune response stimulation against pili and flagella antigens was adequate. Given the high levels of IL5 titer and IgG1 antibody, the stimulated immune response was toward Th1. Humoral immune response stimulation is of key importance in prevention of cholera. Our immunological analysis shows the appropriate immune response in mice model after vaccination with recombinant proteins. The high level of IL5 and low level of IFN-γ show the activation of Th2 cell response. PMID:28191473

  10. Arabidopsis actin capping protein (AtCP) subunits have different expression patterns, and downregulation of AtCPB confers increased thermotolerance of Arabidopsis after heat shock stress.

    PubMed

    Wang, Jue; Qian, Dong; Fan, Tingting; Jia, Honglei; An, Lizhe; Xiang, Yun

    2012-09-01

    As a heterodimer actin-binding protein, capping protein is composed of α and β subunits, and can stabilize the actin filament cytoskeleton by binding to F-actin ends to inhibit G-actin addition or loss from that end. Until now, studies on plant capping protein have focused on biochemical functions in vitro, and so the expression patterns and physiological functions of actin capping protein in Arabidopsis (AtCP) are poorly understood. In the present study, real-time quantitative PCR and Western blot analysis showed that although AtCP α and β subunits (i.e. AtCPA and AtCPB) were expressed in various tissues, their expression patterns were significantly different. GUS staining further indicated they were present in different parts of the same organs. We also demonstrated that the expression levels of both subunits were induced by heat shock stress. However, only the atcpβ-mutant showed enhanced thermotolerance, and confocal microscopy showed that the actin filaments of the atcpβ-mutant were much more complete than that in the wild-type and the atcpα-mutant after heat treatment at 45 °C for 40 and 45 min. In conclusion, these results demonstrated that AtCPA and AtCPB showed distinct expression patterns in vivo, and that downregulation of AtCPB conferred increased plant thermotolerance after heat shock stress.

  11. Molecular Recognition at the Protein-Hydroxyapatite Interface

    SciTech Connect

    Stayton, Partick S.; Drobny, G. P.; Shaw, Wendy J.; Long, Joanna R.; Gilbert, Michelle R.

    2003-09-01

    Proteins found in mineralized tissues act as nature's crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals, such as hydroxyapitite (bones/teeth) and calcium oxalate (kidney stones). Despite their importance in hard-tissue formation and remodeling, and in pathological processes such as stone formation and arterial calcification, there is little known of the protein structure-function relationships that govern hard-tissue engineering. Here we review early studies that have utilized solid-state NMR (ssNMR) techniques to provide in situ secondary-structure determination of statherin and statherin peptides on their biologically relevant hydroxyapatite (HAP) surfaces. In addition to direct structural study, molecular dynamics studies have provided considerable insight into the protein-binding footprint on hydroxyapatite. The molecular insight provided by these studies has also led to the design of biomimetic fusion peptides that utilize nature's crystal-recognition mechanism to display accessible and dynamic bioactive sequences from the HAP surface. These peptides selectively engage adhesion receptors and direct specific outside-in signaling pathway activation in osteoblast-like cells.

  12. Protein Denaturants at Aqueous–Hydrophobic Interfaces: Self-Consistent Correlation between Induced Interfacial Fluctuations and Denaturant Stability at the Interface

    PubMed Central

    2015-01-01

    The notion of direct interaction between denaturing cosolvent and protein residues has been proposed in dialogue relevant to molecular mechanisms of protein denaturation. Here we consider the correlation between free energetic stability and induced fluctuations of an aqueous–hydrophobic interface between a model hydrophobically associating protein, HFBII, and two common protein denaturants, guanidinium cation (Gdm+) and urea. We compute potentials of mean force along an order parameter that brings the solute molecule close to the known hydrophobic region of the protein. We assess potentials of mean force for different relative orientations between the protein and denaturant molecule. We find that in both cases of guanidinium cation and urea relative orientations of the denaturant molecule that are parallel to the local protein–water interface exhibit greater stability compared to edge-on or perpendicular orientations. This behavior has been observed for guanidinium/methylguanidinium cations at the liquid–vapor interface of water, and thus the present results further corroborate earlier findings. Further analysis of the induced fluctuations of the aqueous–hydrophobic interface upon approach of the denaturant molecule indicates that the parallel orientation, displaying a greater stability at the interface, also induces larger fluctuations of the interface compared to the perpendicular orientations. The correlation of interfacial stability and induced interface fluctuation is a recurring theme for interface-stable solutes at hydrophobic interfaces. Moreover, observed correlations between interface stability and induced fluctuations recapitulate connections to local hydration structure and patterns around solutes as evidenced by experiment (Cooper et al., J. Phys. Chem. A2014, 118, 5657.) and high-level ab initio/DFT calculations (Baer et al., Faraday Discuss2013, 160, 89). PMID:25536388

  13. A Metazoan ATAC Acetyltransferase Subunit That Regulates Mitogen-activated Protein Kinase Signaling Is Related to an Ancient Molybdopterin Synthase Component*

    PubMed Central

    Suganuma, Tamaki; Mushegian, Arcady; Swanson, Selene K.; Florens, Laurence; Washburn, Michael P.; Workman, Jerry L.

    2012-01-01

    Molybdopterin (MPT) synthase is an essential enzyme involved in the synthesis of the molybdenum cofactor precursor molybdopterin. The molybdenum cofactor biosynthetic pathway is conserved from prokaryotes to Metazoa. CG10238 is the Drosophila homolog of the MoaE protein, a subunit of MPT synthase, and is found in a fusion with the mitogen-activated protein kinase (MAPK)-upstream protein kinase-binding inhibitory protein (MBIP). This fused protein inhibits the activation of c-Jun N-terminal kinase (JNK). dMoaE (CG10238) carries out this function as a subunit of the ATAC histone acetyltransferase complex. In this study, we demonstrate that Drosophila MoaE (CG10238) also interacts with Drosophila MoaD and with itself to form a complex with stoichiometry identical to the MPT synthase holoenzyme in addition to its function in ATAC. We also show that sequence determinants that regulate MAPK signaling are located within the MoaE region of dMoaE (CG10238). Analysis of other metazoan MBIPs reveals that MBIP protein sequences have an N-terminal region that appears to have been derived from the MoaE protein, although it has lost residues responsible for catalytic activity. Thus, intact and modified copies of the MoaE protein may have been conscripted to play a new, noncatalytic role in MAPK signaling in Metazoa as part of the ATAC complex. PMID:22345504

  14. Sensitive proton-detected solid-state NMR spectroscopy of large proteins with selective CH3 labelling: application to the 50S ribosome subunit

    PubMed Central

    Kurauskas, Vilius; Crublet, Elodie; Macek, Pavel; Kerfah, Rime; Gauto, Diego F.; Boisbouvier, Jérôme; Schanda, Paul

    2016-01-01

    Solid-state NMR spectroscopy allows the characterization of structure, interactions and dynamics of insoluble and/or very large proteins. Sensitivity and resolution are often major challenges for obtaining atomic-resolution information, in particular for very large protein complexes. Here we show that the use of deuterated, specifically CH3-labelled proteins result in significant sensitivity gains compared to previously employed CHD2 labelling, while line widths only marginally increase. We apply this labelling strategy to a 468 kDa-large dodecameric aminopeptidase, TET2, and the 1.6 MDa-large 50S ribosome subunit of Thermus thermophilus. PMID:27385633

  15. Swiss Cheese, a protein involved in progressive neurodegeneration acts as a non-canonical regulatory subunit for PKA-C3

    PubMed Central

    da Cruz, Alexandre Bettencourt; Wentzell, Jill; Kretzschmar, Doris

    2008-01-01

    The Drosophila Swiss Cheese (SWS) protein and its vertebrate orthologue Neuropathy Target Esterase (NTE) are required for neuronal survival and glial integrity. In humans, NTE is the target of organophosphorous compounds which cause a paralyzing axonal degeneration and recently mutations in NTE have been shown to cause a Hereditary Spastic Paraplegia called NTE-related Motor-Neuron Disorder. SWS and NTE are concentrated in the endoplasmic reticulum and both have been shown to have an esterase function against an artificial substrate. However, the functional mechanisms and the pathways in which SWS/NTE are involved in are still widely unknown. Here we show that SWS interacts specifically with the C3 catalytic subunit of cAMP activated protein kinase (PKA-C3) which together with orthologues in mouse (Pkare) and human (PrKX) forms a novel class of catalytic subunits of unknown function. This interaction requires a domain of SWS which shows homology to regulatory subunits of PKA and, like conventional regulatory subunits, the binding of SWS to the PKA-C3 inhibits is function. Consistent with this result, expression of additional PKA-C3 induces degeneration and enhances the neurodegenerative phenotype in sws mutants. We also show that the complex formation with the membrane-bound SWS tethers PKA-C3 to membranes. We therefore propose a model in which SWS acts as a non-canonical subunit for PKA-C3, whereby the complex formation regulates the localization and kinase activity of PKA-C3, and that disruption of this regulation can induce neurodegeneration. PMID:18945896

  16. G-protein Stimulatory α Subunit Is Involved in Osteogenic Activity in Osteoblastic Cell Line SaOS-2 Cells.