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Posttranslational modification of CENP-A influences the conformation of centromeric chromatin.
Bailey AO, Panchenko T, Sathyan KM, Petkowski JJ, Pai PJ, Bai DL, Russell DH, Macara IG, Shabanowitz J, Hunt DF, Black BE, Foltz DR
(2013) Proc Natl Acad Sci U S A 110: 11827-32
MeSH Terms: Autoantigens, Cell Cycle Proteins, Cell Line, Centromere, Centromere Protein A, Chromatin, Chromatography, High Pressure Liquid, Chromosomal Proteins, Non-Histone, Epigenesis, Genetic, Guanine Nucleotide Exchange Factors, Humans, Mass Spectrometry, Methylation, Molecular Conformation, Nuclear Proteins, Phosphorylation, Protein Processing, Post-Translational, Ultracentrifugation
Show Abstract · Added March 7, 2014
Centromeres are chromosomal loci required for accurate segregation of sister chromatids during mitosis. The location of the centromere on the chromosome is not dependent on DNA sequence, but rather it is epigenetically specified by the histone H3 variant centromere protein A (CENP-A). The N-terminal tail of CENP-A is highly divergent from other H3 variants. Canonical histone N termini are hotspots of conserved posttranslational modification; however, no broadly conserved modifications of the vertebrate CENP-A tail have been previously observed. Here, we report three posttranslational modifications on human CENP-A N termini using high-resolution MS: trimethylation of Gly1 and phosphorylation of Ser16 and Ser18. Our results demonstrate that CENP-A is subjected to constitutive initiating methionine removal, similar to other H3 variants. The nascent N-terminal residue Gly1 becomes trimethylated on the α-amino group. We demonstrate that the N-terminal RCC1 methyltransferase is capable of modifying the CENP-A N terminus. Methylation occurs in the prenucleosomal form and marks the majority of CENP-A nucleosomes. Serine 16 and 18 become phosphorylated in prenucleosomal CENP-A and are phosphorylated on asynchronous and mitotic nucleosomal CENP-A and are important for chromosome segregation during mitosis. The double phosphorylation motif forms a salt-bridged secondary structure and causes CENP-A N-terminal tails to form intramolecular associations. Analytical ultracentrifugation of phospho-mimetic CENP-A nucleosome arrays demonstrates that phosphorylation results in greater intranucleosome associations and counteracts the hyperoligomerized state exhibited by unmodified CENP-A nucleosome arrays. Our studies have revealed that the major modifications on the N-terminal tail of CENP-A alter the physical properties of the chromatin fiber at the centromere.
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18 MeSH Terms
In vitro uncoating of HIV-1 cores.
Shah VB, Aiken C
(2011) J Vis Exp :
MeSH Terms: Capsid, Cell Line, Centrifugation, Density Gradient, HIV-1, Humans, Sucrose, Ultracentrifugation, Virion, Virology
Show Abstract · Added February 19, 2015
The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone(1, 2). Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core. Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form(3) in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells(4). This indicates that the stability of the capsid is critical for HIV-1 infection. HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37°C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability(4, 5, 6). It should also be useful for studying the role of cellular factors in HIV-1 uncoating.
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9 MeSH Terms
Nanostructural and transcriptomic analyses of human saliva derived exosomes.
Palanisamy V, Sharma S, Deshpande A, Zhou H, Gimzewski J, Wong DT
(2010) PLoS One 5: e8577
MeSH Terms: Blotting, Western, Exosomes, Gene Expression Profiling, Humans, Microscopy, Atomic Force, Nanostructures, Saliva, Ultracentrifugation
Show Abstract · Added October 15, 2011
BACKGROUND - Exosomes, derived from endocytic membrane vesicles are thought to participate in cell-cell communication and protein and RNA delivery. They are ubiquitous in most body fluids (breast milk, saliva, blood, urine, malignant ascites, amniotic, bronchoalveolar lavage, and synovial fluids). In particular, exosomes secreted in human saliva contain proteins and nucleic acids that could be exploited for diagnostic purposes. To investigate this potential use, we isolated exosomes from human saliva and characterized their structural and transcriptome contents.
METHODOLOGY - Exosomes were purified by differential ultracentrifugation and identified by immunoelectron microscopy (EM), flow cytometry, and Western blot with CD63 and Alix antibodies. We then described the morphology, shape, size distribution, and density using atomic force microscopy (AFM). Microarray analysis revealed that 509 mRNA core transcripts are relatively stable and present in the exosomes. Exosomal mRNA stability was determined by detergent lysis with RNase A treatment. In vitro, fluorescently labeled saliva exosomes could communicate with human keratinocytes, transferring their genetic information to human oral keratinocytes to alter gene expression at a new location.
CONCLUSION - Our findings are consistent with the hypothesis that exosomes shuttle RNA between cells and that the RNAs present in the exosomes may be a possible resource for disease diagnostics.
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8 MeSH Terms
Apolipoprotein AI tertiary structures determine stability and phospholipid-binding activity of discoidal high-density lipoprotein particles of different sizes.
Chen B, Ren X, Neville T, Jerome WG, Hoyt DW, Sparks D, Ren G, Wang J
(2009) Protein Sci 18: 921-35
MeSH Terms: Apolipoprotein A-I, Cholates, Dialysis, Escherichia coli, Humans, Lipoproteins, Lipoproteins, HDL, Microscopy, Electron, Nuclear Magnetic Resonance, Biomolecular, Particle Size, Phospholipids, Protein Binding, Protein Stability, Protein Structure, Tertiary, Recombinant Proteins, Ultracentrifugation
Show Abstract · Added December 10, 2013
Human high-density lipoprotein (HDL) plays a key role in the reverse cholesterol transport pathway that delivers excess cholesterol back to the liver for clearance. In vivo, HDL particles vary in size, shape and biological function. The discoidal HDL is a 140-240 kDa, disk-shaped intermediate of mature HDL. During mature spherical HDL formation, discoidal HDLs play a key role in loading cholesterol ester onto the HDL particles by activating the enzyme, lecithin:cholesterol acyltransferase (LCAT). One of the major problems for high-resolution structural studies of discoidal HDL is the difficulty in obtaining pure and, foremost, homogenous sample. We demonstrate here that the commonly used cholate dialysis method for discoidal HDL preparation usually contains 5-10% lipid-poor apoAI that significantly interferes with the high-resolution structural analysis of discoidal HDL using biophysical methods. Using an ultracentrifugation method, we quickly removed lipid-poor apoAI. We also purified discoidal reconstituted HDL (rHDL) into two pure discoidal HDL species of different sizes that are amendable for high-resolution structural studies. A small rHDL has a diameter of 7.6 nm, and a large rHDL has a diameter of 9.8 nm. We show that these two different sizes of discoidal HDL particles display different stability and phospholipid-binding activity. Interestingly, these property/functional differences are independent from the apoAI alpha-helical secondary structure, but are determined by the tertiary structural difference of apoAI on different discoidal rHDL particles, as evidenced by two-dimensional NMR and negative stain electron microscopy data. Our result further provides the first high-resolution NMR data, demonstrating a promise of structural determination of discoidal HDL at atomic resolution using a combination of NMR and other biophysical techniques.
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16 MeSH Terms
A pathway-dependent on apoE, ApoAI, and ABCA1 determines formation of buoyant high-density lipoprotein by macrophage foam cells.
Yancey PG, Yu H, Linton MF, Fazio S
(2007) Arterioscler Thromb Vasc Biol 27: 1123-31
MeSH Terms: ATP Binding Cassette Transporter 1, ATP-Binding Cassette Transporters, Animals, Apolipoprotein A-I, Apolipoproteins E, Atherosclerosis, Cell Count, Cholesterol, Disease Progression, Female, Foam Cells, Mice, Mice, Inbred C57BL, Ultracentrifugation
Show Abstract · Added December 10, 2013
OBJECTIVE - ABCA1-dependent and ABCA1-independent pathways may operate in high-density lipoprotein formation by macrophages secreting apolipoprotein (apo) E. We examined the impact of ABCA1 on apoE-mediated efflux from cholesterol-enriched macrophages.
METHODS AND RESULTS - Without acceptors, wild-type, ABCA1-/-, and apoE-/- macrophages released 5.7%+/-0.3%, 1.8%+/-0.1%, and 2.3%+/-0.2% of their cholesterol, and the LXR agonist, TO-901317, enhanced efflux by 137%, 10%, and 20%. Although similar amounts of apoE were secreted from ABCA1-/- and wild-type cells, apoE from ABCA1-/- cells was only partially phospholipidated and floated at density > 1.21 g/mL, whereas apoE from wild-type cells floated at density of 1.09 to 1.17 g/mL and paralleled the density of cholesterol. With apoAI, LXR stimulation increased efflux by 139% and 86% from wild-type and apoE-/- cells, resulting in a large difference in efflux (29.5%+/-0.2% versus 17.0%+/-0.5%). The density of apoE and cholesterol from wild-type cells did not change with apoAI, and most apoAI floated at density > or = 1.17 g/mL. In apoE-/- cells, apoAI and cholesterol floated at similar density, but the peak fraction only contained 4 microg cholesterol/mg protein versus 18 in WT cells.
CONCLUSIONS - Macrophage apoE requires ABCA1 for formation of high-density lipoprotein. ApoAI facilitates association of apoE with more buoyant high-density lipoprotein, suggesting that apoE, plasma apoAI, and ABCA1 operate together to optimize mobilization of macrophage cholesterol, a process critical to limiting plaque development.
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14 MeSH Terms
Plasma insulin levels predict atherosclerotic lesion burden in obese hyperlipidemic mice.
Gruen ML, Saraswathi V, Nuotio-Antar AM, Plummer MR, Coenen KR, Hasty AH
(2006) Atherosclerosis 186: 54-64
MeSH Terms: Animals, Atherosclerosis, Biomarkers, Cholesterol, HDL, Cholesterol, LDL, Cholesterol, VLDL, Disease Models, Animal, Gas Chromatography-Mass Spectrometry, Hyperlipidemias, Insulin, Insulin Resistance, Mice, Mice, Inbred C57BL, Obesity, Prognosis, Severity of Illness Index, Ultracentrifugation
Show Abstract · Added December 5, 2013
Despite a clear association between obesity, insulin resistance and atherosclerosis in humans, to date, no animal models have been described in which insulin resistance is associated with atherosclerotic lesion burden. Using two mouse models of obesity-induced hyperlipidemia:leptin deficient (ob/ob) mice on an apolipoprotein E deficient (apoE-/-) or low density lipoprotein receptor deficient (LDLR-/-) background, we sought to determine metabolic parameters most closely associated with atherosclerotic lesion burden. Total plasma cholesterol (TC) levels in ob/ob;apoE-/- mice and ob/ob;LDLR-/- mice were indistinguishable (682+/-48 versus 663+/-16, respectively). Analysis of lipoprotein profiles showed that cholesterol was carried primarily on VLDL in the ob/ob;apoE-/- mice and on LDL in the ob/ob;LDLR-/- mice. Plasma triglycerides (TG) were 55% lower (P<0.001), non-esterified fatty acids (NEFA) were 1.5-fold higher (P<0.01), and insulin levels were 1.7-fold higher (NS) in ob/ob;apoE-/- mice compared to ob/ob;LDLR-/- mice. Other parameters such as body weight, fat pad weight, and glucose levels were not different between the groups. Aortic sinus lesion area of ob/ob;apoE-/- mice was increased 3.2-fold above ob/ob;LDLR-/- mice (102,455+/-8565 microm2/section versus 31,750+/-4478 microm2/section, P<0.001). Lesions in ob/ob;apoE-/- mice were also more complex as evidenced by a 7.7-fold increase in collagen content (P<0.001). Atherosclerotic lesion area was positively correlated with body weight (P<0.005), NEFA (P=0.007), and insulin (P=0.002) levels in the ob/ob;LDLR-/- mice and with insulin (P=0.014) in the ob/ob;apoE-/- mice. In contrast, lesion burden was neither associated with TC and TG, nor with individual lipoprotein pools, in either animal model. These data provide a direct demonstration of the pathophysiologic relevance of hyperinsulinemia, NEFA, and increased body weight to atherosclerotic lesion formation.
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17 MeSH Terms
Structural and functional analysis of essential pre-mRNA splicing factor Prp19p.
Ohi MD, Vander Kooi CW, Rosenberg JA, Ren L, Hirsch JP, Chazin WJ, Walz T, Gould KL
(2005) Mol Cell Biol 25: 451-60
MeSH Terms: Cell Cycle Proteins, Chromatography, Gel, Circular Dichroism, DNA Mutational Analysis, Dimerization, Genotype, Image Processing, Computer-Assisted, Immunoblotting, Immunoprecipitation, Microscopy, Electron, Models, Biological, Polymerase Chain Reaction, Protein Binding, Protein Conformation, Protein Structure, Tertiary, RNA Splicing, RNA Splicing Factors, RNA, Messenger, RNA-Binding Proteins, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Schizosaccharomyces, Schizosaccharomyces pombe Proteins, Spliceosomes, Structure-Activity Relationship, Temperature, Two-Hybrid System Techniques, Ultracentrifugation
Show Abstract · Added December 10, 2013
U-box-containing Prp19p is an integral component of the Prp19p-associated complex (the nineteen complex, or NTC) that is essential for activation of the spliceosome. Prp19p makes numerous protein-protein contacts with other NTC components and is required for NTC stability. Here we show that Prp19p forms a tetramer in vitro and in vivo and we map the domain required for its oligomerization to a central tetrameric coiled-coil. Biochemical and in vivo analyses are consistent with Prp19p tetramerization providing an interaction surface for a single copy of its binding partner, Cef1p. Electron microscopy showed that the isolated Prp19p tetramer is an elongated particle consisting of four globular WD40 domains held together by a central stalk consisting of four N-terminal U-boxes and four coiled-coils. These structural and functional data provide a basis for understanding the role of Prp19p as a key architectural component of the NTC.
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29 MeSH Terms
Structure formation in the C terminus of type III collagen guides disulfide cross-linking.
Boudko SP, Engel J
(2004) J Mol Biol 335: 1289-97
MeSH Terms: Calorimetry, Differential Scanning, Circular Dichroism, Collagen Type III, Cross-Linking Reagents, Disulfides, Escherichia coli, Mass Spectrometry, Oxidation-Reduction, Peptide Fragments, Protein Conformation, Protein Folding, Recombinant Proteins, Ultracentrifugation, Viral Proteins
Show Abstract · Added November 2, 2017
In type III collagen the main triple-helical domain is followed by a disulfide knot and the C-terminal propeptide, which are both essential for nucleation, stabilization and registration of the triple helix. We demonstrate that oxidative inter-chain disulfide bridging does not occur between the knot sequences GlyProCysCysGly of dissociated randomly coiled chains. N-terminal fusion of the obligatory trimeric domain of mini-fibritin is able to direct this process efficiently, demonstrating a folded precursor mechanism in which the thiol groups have to be properly placed for the formation of native disulfide bonds. The natural C-propeptide domain may act in a similar way as the mini-fibritin domain. After disulfide linkage and triple-helix formation the catalyzing mini-fibritin domain was removed by thrombin cleavage. In this way a short but stable triple-helical collagen fragment was expressed in Escherichia coli for structural and functional studies.
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14 MeSH Terms
Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins.
Shi M, Bennett TA, Cimino DF, Maestas DC, Foutz TD, Gurevich VV, Sklar LA, Prossnitz ER
(2003) Biochemistry 42: 7283-93
MeSH Terms: Animals, Arrestin, Calcium, GTP-Binding Protein alpha Subunit, Gi2, GTP-Binding Protein alpha Subunits, Gi-Go, GTP-Binding Proteins, Heterotrimeric GTP-Binding Proteins, Humans, Leukemia, Myeloid, Mice, Microscopy, Confocal, N-Formylmethionine Leucyl-Phenylalanine, Phosphorylation, Protein Subunits, Proto-Oncogene Proteins, Receptors, Formyl Peptide, Receptors, Immunologic, Receptors, Peptide, Recombinant Fusion Proteins, Transfection, Tumor Cells, Cultured, U937 Cells, Ultracentrifugation
Show Abstract · Added December 10, 2013
G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.
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23 MeSH Terms
Collagen triple helix formation can be nucleated at either end.
Frank S, Boudko S, Mizuno K, Schulthess T, Engel J, Bächinger HP
(2003) J Biol Chem 278: 7747-50
MeSH Terms: Amino Acid Sequence, Collagen, Kinetics, Mass Spectrometry, Molecular Sequence Data, Protein Conformation, Protein Denaturation, Protein Folding, Recombinant Proteins, Ultracentrifugation
Show Abstract · Added November 2, 2017
The directional dependence of folding rates for rod-like macromolecules such as parallel alpha-helical coiled-coils, DNA double-helices, and collagen triple helices is largely unexplored. This is mainly due to technical difficulties in measuring rates in different directions. Folding of collagens is nucleated by trimeric non-collagenous domains. These are usually located at the COOH terminus, suggesting that triple helix folding proceeds from the COOH to the NH(2) terminus. Evidence is presented here that effective nucleation is possible at both ends of the collagen-like peptide (Gly-Pro-Pro)(10), using designed proteins in which this peptide is fused either NH(2)- or COOH-terminal to a nucleation domain, either T4-phage foldon or the disulfide knot of type III collagen. The location of the nucleation domain influences triple-helical stability, which might be explained by differences in the linker sequences and the presence or absence of repulsive charges at the carboxyl-terminal end of the triple helix. Triple helical folding rates are found to be independent of the site of nucleation and consistent with cis-trans isomerization being the rate-limiting step.
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10 MeSH Terms