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PubMed2005

Characterization of the human GARP (Golgi associated retrograde protein) complex.

The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the "orphan" SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells.

Animals↗
PubMed2006

Analysis of Nedd8-associated polypeptides: a model for deciphering the pathway for ubiquitin-like modifications.

Ubiquitin-like proteins modify target proteins, altering their activities or causing them to be slated for degradation. These modifications are used to efficiently regulate key events in the cell. To explore the set of proteins modified by a small ubiquitin-like protein, we have developed a proteomic approach. Affinity purification of an epitope-tagged Nedd8 allowed the identification of the majority of proteins known to be involved with the neddylation pathway. This purification not only isolated the known targets of neddylation but also the constellation of enzymes and complexes known to regulate neddylation and deneddylation, including the COP9 signalosome, Nub1, and enzymes in the neddylation cascade. This purification scheme can be applied to other small ubiquitin-like proteins, especially those with limited protein targets such as the SUMOs (1, 2, and 3), Isg15, or FAT10.

COP9 Signalosome Complex↗
PubMed2004

Optimized Baxter model of protein solutions: electrostatics versus adhesion.

A theory is set up of spherical proteins interacting by screened electrostatics and constant adhesion, in which the effective adhesion parameter is optimized by a variational principle for the free energy. An analytical approach to the second virial coefficient is first outlined by balancing the repulsive electrostatics against part of the bare adhesion. A theory similar in spirit is developed at nonzero concentrations by assuming an appropriate Baxter model as the reference state. The first-order term in a functional expansion of the free energy is set equal to zero which determines the effective adhesion as a function of salt and protein concentrations. The resulting theory is shown to have fairly good predictive power for the ionic-strength dependence of both the second virial coefficient and the osmotic pressure or compressibility of lysozyme up to about 0.2 volume fraction.

Adhesiveness↗
PubMed2005

The single chlorophyll a molecule in the cytochrome b6f complex: unusual optical properties protect the complex against singlet oxygen.

The cytochrome b(6)f complex of oxygenic photosynthesis mediates electron transfer between the reaction centers of photosystems I and II and facilitates coupled proton translocation across the membrane. High-resolution x-ray crystallographic structures (Kurisu et al., 2003; Stroebel et al., 2003) of the cytochrome b(6)f complex unambiguously show that a Chl a molecule is an intrinsic component of the cytochrome b(6)f complex. Although the functional role of this Chl a is presently unclear (Kuhlbrandt, 2003), an excited Chl a molecule is known to produce toxic singlet oxygen as the result of energy transfer from the excited triplet state of the Chl a to oxygen molecules. To prevent singlet oxygen formation in light-harvesting complexes, a carotenoid is typically positioned within approximately 4 A of the Chl a molecule, effectively quenching the triplet excited state of the Chl a. However, in the cytochrome b(6)f complex, the beta-carotene is too far (> or =14 Angstroms) from the Chl a for effective quenching of the Chl a triplet excited state. In this study, we propose that in this complex, the protection is at least partly realized through special arrangement of the local protein structure, which shortens the singlet excited state lifetime of the Chl a by a factor of 20-25 and thus significantly reduces the formation of the Chl a triplet state. Based on optical ultrafast absorption difference experiments and structure-based calculations, it is proposed that the Chl a singlet excited state lifetime is shortened due to electron exchange transfer with the nearby tyrosine residue. To our knowledge, this kind of protection mechanism against singlet oxygen has not yet been reported for any other chlorophyll-containing protein complex. It is also reported that the Chl a molecule in the cytochrome b(6)f complex does not change orientation in its excited state.

Biophysical Phenomena↗
PubMed2004

The COP9 signalosome: mediating between kinase signaling and protein degradation.

The COP9 Signalosome (CSN), a highly conserved eight-subunit complex, is found in all higher eukaryotes. It contains eight core subunits, named CSN1-8, in order of decreasing molecular weight. The CSN is structurally similar to the regulatory lid of 26S proteasome and the eukaryotic translation initiation factor eIF3. CSN is also now known to play an essential role in signaling processes controlling many aspects of plant and Drosophila development. Taken together, the various genetic studies demonstrate that the CSN is involved at the nexus between multiple signal inputs and a variety of downstream regulatory cascades controlling specific aspects of cellular differentiation. Research in various organisms has converged onto the notion that CSN is biochemically linked to ubiquitin-dependent protein degradation. Other proposed roles for the CSN include regulating eIF3 and kinase signaling. CSN is itself is both a target for kinase activity and associates with and coordinates activity of kinases. CSN-associated kinases. This kinase activity further regulates the ubiquitin-dependent degradation of various transcription factors. This review concentrates on the proposed activity of the CSN as a regulator of protein phosphorylation.

Animals↗
PubMed2004

Plant immunity: the origami of receptor activation.

Mutations in plant cytosolic HSP90 genes have been found to impair the immune responses triggered by host pathogen receptors. HSP90 links the plant receptors to other components essential for receptor function. The new findings suggest mechanistic parallels with steroid receptor regulation in animals.

COP9 Signalosome Complex↗
PubMed2001

Spatial regulation of the exocyst complex by Rho1 GTPase.

Spatial regulation of membrane traffic is fundamental to many biological processes, including epithelial cell polarization and neuronal synaptogenesis. The multiprotein exocyst complex is localized to sites of polarized exocytosis, and is required for vesicle targeting and docking at specific domains of the plasma membrane. One component of the complex, Sec3, is thought to be a spatial landmark for polarized exocytosis. We have searched for proteins that regulate the polarized localization of the exocyst in the budding yeast Saccharomyces cerevisiae. Here we report that certain rho1 mutant alleles specifically affect the localization of the exocyst proteins. Sec3 interacts directly with Rho1 in its GTP-bound form, and functional Rho1 is needed both to establish and to maintain the polarized localization of Sec3. Sec3 is not the only mediator of the effect of Rho1 on the exocyst, because some members of the complex are correctly targeted independently of the interaction between Rho1 and Sec3. These results reveal the action of parallel pathways for the polarized localization of the exocytic machinery, both of which are under the control of Rho1, a master regulator of cell polarity.

Binding Sites↗
PubMed1995

The 68 kDa calmodulin-binding protein is tightly associated with the multiprotein DNA polymerase alpha-primase complex in HeLa cells.

Calcium and its receptor protein calmodulin function in the regulation of proliferation of mammalian cells. A 68 kDa calmodulin-specific binding protein was shown previously to be associated with growth factor-dependent progression of a variety of mammalian cells from G1 to S phase and to stimulate DNA synthesis in permeabilized hematopoietic progenitor cells. In this report we show that the 68 kDa calmodulin-specific binding protein in HeLa cells is tightly associated with the DNA polymerase alpha-primase component of the 21S complex of enzymes for DNA synthesis. The 68 kDa calmodulin-binding protein and the DNA polymerase alpha-primase complex cofractionate during Q-Sepharose chromatography to isolate the 21S enzyme complex, native and denatured DNA-cellulose to dissociate the 21S complex, and DEAE-Bio-Gel chromatography to isolate the multiprotein DNA polymerase alpha-primase complex. The 68 kDa calmodulin-specific binding protein and DNA polymerase alpha also bind and coelute during affinity chromatography on calmodulin-agarose. They also coprecipitate with C10-agarose-linked monoclonal antibody SJK 132-20 to human DNA polymerase alpha. The tight association of the 68 kDa calmodulin-binding protein to the DNA polymerase alpha-primase complex supports a function for this protein in the regulation of DNA synthesis in vivo.

Calmodulin↗
PubMed1999

Evidence for evolutionary conservation of a physical linkage between the human BAF60b, a subunit of SWI/SNF complex, and thyroid hormone receptor interacting protein-1 genes on chromosome 17.

The ubiquitously expressed rat BAF60b gene, which codes for a subunit of the multiprotein SWI/SNF complex, was recently identified between the pituitary growth hormone (GH-N) and thyroid hormone receptor interacting protein-1 (TRIP-1) genes. In primates, duplication of the GH-N gene has resulted in the addition of four placenta-specific (GH-V, CS-A, CS-B, and CS-L) genes downstream of the GH-N gene. As part of our study of the effect of remote sequences on the transcriptional regulation of the GH/CS gene family, we showed recently that these genes lie 40 kb upstream of the human TRIP-1 gene. We have now investigated the presence of the human BAF60b gene upstream of the TRIP-1 gene for evidence of evolutionary conservation of this arrangement or its disruption by the recent duplication of the nearby GH-N gene in primates. We report that, as in the rat genome, the human BAF60b gene is in the reverse transcriptional direction relative to the TRIP-1 gene, such that their polyadenylation sites are separated by 93 bp which compares with 92 bp in the rat. Reexamination of reported porcine TRIP-1 sequences also revealed the presence of the BAF60b gene separated by 93 bp, supporting an evolutionary conservation of this arrangement.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2005

CAND1 enhances deneddylation of CUL1 by COP9 signalosome.

Cullin-RING ligases (CRLs) regulate diverse cellular functions such as cell cycle progression and cytokine signaling by ubiquitinating key regulatory proteins. The activity of CRLs is controlled by Nedd8 modification of the cullin subunits. Recent reports have suggested that CAND1, which specifically binds to unmodified CUL1 but not to neddylated one, is required for the in vivo function of SCFs, the CUL1-containing CRLs. We show here that CAND1 and COP9 signalosome (CSN), the major deneddylase of cullins, bind to unneddylated CUL1 in a mutually exclusive way. The suppression of CAND1 expression by small inhibitory RNA enhanced the interaction between CUL1 and CSN, suggesting that CAND1 inhibited the binding of CSN to CUL1. We found that the binding of CSN to CUL1 required the four helix bundle in CUL1 C-terminal domain, which was wrapped around by CAND1 in the CAND1-CUL1-Rbx1 complex. CAND1 greatly facilitated CSN-mediated deneddylation of CUL1 in vitro, which was dependent on its binding to CUL1. Our data suggest that enhancement of CSN-mediated deneddylation by CAND1 may contribute to its function as a positive regulator of SCFs in vivo.

COP9 Signalosome Complex↗
PubMed2006

WAVE signalling: from biochemistry to biology.

The small GTPases Rho, Rac and Cdc42 (cell-division cycle 42) function as molecular switches to modulate the actin cytoskeleton. They achieve this by modulating the activity of downstream cellular targets. One group of Rho GTPase effectors, WAVE (Wiskott-Aldrich syndrome protein verprolin homologous)-1, WAVE-2 and WAVE-3, function as scaffolds for actin-based signalling complexes. The present review highlights current knowledge regarding the biochemistry of the WAVE signalling complexes and their biological significance.

Animals↗
PubMed2006

Expression, purification and crystallization of a PCI domain from the COP9 signalosome subunit 7 (CSN7).

A core fragment of Arabidopsis thaliana COP9 signalosome (CSN) subunit 7 was expressed in Escherichia coli. The protein was purified to homogeneity and screened for crystallization. Crystallization conditions were refined using the sitting-drop vapour-diffusion method. Crystals were obtained using polyethylene glycol 8000 as a precipitant and have a thick rod-like morphology. Their crystallographic symmetry is orthorhombic, space group C222(1), with unit-cell parameters a = 57.2, b = 86.2, c = 72.6 A and a diffraction limit of 2.06 A.

Arabidopsis↗
PubMed2006

Ubiquitin and NEDD8: brothers in arms.

Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins are small signaling molecules that are involved in many aspects of cell function. It has been assumed that Ub and Ubl have functionally distinct roles because they use different conjugation machineries and bind to different effector proteins. This paradigm, however, must be revisited after recent findings that signaling cascades mediated by Ub and the Ubl NEDD8 (Neural precursor cell-Expressed Developmentally Down-regulated 8) in the regulation of epidermal growth factor receptor (EGFR) endocytosis are redundant. In this context, Ub and NEDD8 share the same E3 ligase, Cbl, and are recognized by identical components of the endocytic sorting machinery. This unexpected redundancy introduces additional complexity to the current view of Ub signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Structural insights into SRP RNA: an induced fit mechanism for SRP assembly.

Proper assembly of large protein-RNA complexes requires sequential binding of the proteins to the RNA. The signal recognition particle (SRP) is a multiprotein-RNA complex responsible for the cotranslational targeting of proteins to biological membranes. Here we describe the crystal structure at 2.6-A resolution of the S-domain of SRP RNA from the archeon Methanococcus jannaschii. Comparison of this structure with the SRP19-bound form reveals the nature of the SRP19-induced conformational changes, which promote subsequent SRP54 attachment. These structural changes are initiated at the SRP19 binding site and transmitted through helix 6 to looped-out adenosines, which form tertiary RNA interaction with helix 8. Displacement of these adenosines enforces a conformational change of the asymmetric loop structure in helix 8. In free RNA, the three unpaired bases A195, C196, and C197 are directed toward the helical axis, whereas upon SRP19 binding the loop backbone inverts and the bases are splayed out in a conformation that resembles the SRP54-bound form. Nucleotides adjacent to the bulged nucleotides seem to be particularly important in the regulation of this loop transition. Binding of SRP19 to 7S RNA reveals an elegant mechanism of how protein-induced changes are directed through an RNA molecule and may relate to those regulating the assembly of other RNPs.

Adenosine↗
PubMed2005

Copper-dependent inhibition of human cytochrome c oxidase by a dimeric conformer of amyloid-beta1-42.

In studies of Alzheimer's disease pathogenesis there is an increasing focus on mechanisms of intracellular amyloid-beta (Abeta) generation and toxicity. Here we investigated the inhibitory potential of the 42 amino acid Abeta peptide (Abeta1-42) on activity of electron transport chain enzyme complexes in human mitochondria. We found that synthetic Abeta1-42 specifically inhibited the terminal complex cytochrome c oxidase (COX) in a dose-dependent manner that was dependent on the presence of Cu2+ and specific "aging" of the Abeta1-42 solution. Maximal COX inhibition occurred when using Abeta1-42 solutions aged for 3-6 h at 30 degrees C. The level of Abeta1-42-mediated COX inhibition increased with aging time up to approximately 6 h and then declined progressively with continued aging to 48 h. Photo-induced cross-linking of unmodified proteins followed by SDS-PAGE analysis revealed dimeric Abeta as the only Abeta species to provide significant temporal correlation with the observed COX inhibition. Analysis of brain and liver from an Alzheimer's model mouse (Tg2576) revealed abundant Abeta immunoreactivity within the brain mitochondria fraction. Our data indicate that endogenous Abeta is associated with brain mitochondria and that Abeta1-42, possibly in its dimeric conformation, is a potent inhibitor of COX, but only when in the presence of Cu2+. We conclude that Cu2+-dependent Abeta-mediated inhibition of COX may be an important contributor to the neurodegeneration process in Alzheimer's disease.

Amyloid beta-Peptides↗
PubMed2005

Coarse-grained strategy for modeling protein stability in concentrated solutions.

We present a coarse-grained approach for modeling the thermodynamic stability of single-domain globular proteins in concentrated aqueous solutions. Our treatment derives effective protein-protein interactions from basic structural and energetic characteristics of the native and denatured states. These characteristics, along with the intrinsic (i.e., infinite dilution) thermodynamics of folding, are calculated from elementary sequence information using a heteropolymer collapse theory. We integrate this information into Reactive Canonical Monte Carlo simulations to investigate the connections between protein sequence hydrophobicity, protein-protein interactions, protein concentration, and the thermodynamic stability of the native state. The model predicts that sequence hydrophobicity can affect how protein concentration impacts native-state stability in solution. In particular, low hydrophobicity proteins are primarily stabilized by increases in protein concentration, whereas high hydrophobicity proteins exhibit richer nonmonotonic behavior. These trends appear qualitatively consistent with the available experimental data. Although factors such as pH, salt concentration, and protein charge are also important for protein stability, our analysis suggests that some of the nontrivial experimental trends may be driven by a competition between destabilizing hydrophobic protein-protein attractions and entropic crowding effects.

Binding Sites↗
PubMed2006

Specificity of molecular interactions in transient protein-protein interaction interfaces.

In this study, we investigate what types of interactions are specific to their biological function, and what types of interactions are persistent regardless of their functional category in transient protein-protein heterocomplexes. This is the first approach to analyze protein-protein interfaces systematically at the molecular interaction level in the context of protein functions. We perform systematic analysis at the molecular interaction level using classification and feature subset selection technique prevalent in the field of pattern recognition. To represent the physicochemical properties of protein-protein interfaces, we design 18 molecular interaction types using canonical and noncanonical interactions. Then, we construct input vector using the frequency of each interaction type in protein-protein interface. We analyze the 131 interfaces of transient protein-protein heterocomplexes in PDB: 33 protease-inhibitors, 52 antibody-antigens, 46 signaling proteins including 4 cyclin dependent kinase and 26 G-protein. Using kNN classification and feature subset selection technique, we show that there are specific interaction types based on their functional category, and such interaction types are conserved through the common binding mechanism, rather than through the sequence or structure conservation. The extracted interaction types are C(alpha)-- H...O==C interaction, cation...anion interaction, amine...amine interaction, and amine...cation interaction. With these four interaction types, we achieve the classification success rate up to 83.2% with leave-one-out cross-validation at k = 15. Of these four interaction types, C(alpha)--H...O==C shows binding specificity for protease-inhibitor complexes, while cation-anion interaction is predominant in signaling complexes. The amine ... amine and amine...cation interaction give a minor contribution to the classification accuracy. When combined with these two interactions, they increase the accuracy by 3.8%. In the case of antibody-antigen complexes, the sign is somewhat ambiguous. From the evolutionary perspective, while protease-inhibitors and sig-naling proteins have optimized their interfaces to suit their biological functions, antibody-antigen interactions are the happenstance, implying that antibody-antigen complexes do not show distinctive interaction types. Persistent interaction types such as pi...pi, amide-carbonyl, and hydroxyl-carbonyl interaction, are also investigated. Analyzing the structural orientations of the pi...pi stacking interactions, we find that herringbone shape is a major configuration in transient protein-protein interfaces. This result is different from that of protein core, where parallel-displaced configurations are the major configuration. We also analyze overall trend of amide-carbonyl and hydroxyl-carbonyl interactions. It is noticeable that nearly 82% of the interfaces have at least one hydroxyl-carbonyl interactions.

Animals↗