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Darren E. Casteel Shunhui Zhuang Ying Zeng Fred W. Perrino Gerry R. Boss Mehran Goulian Renate B. Pilz 《The Journal of biological chemistry》2009,284(9):5807-5818
α-Accessory factor (AAF) stimulates the activity of DNA
polymerase-α·primase, the only enzyme known to initiate DNA
replication in eukaryotic cells (Goulian, M., Heard, C. J.,
and Grimm, S. L. (1990) J. Biol. Chem. 265
,13221
-13230). We purified the AAF
heterodimer composed of 44- and 132-kDa subunits from cultured cells and
identified full-length cDNA clones using amino acid sequences from internal
peptides. AAF-132 demonstrated no homologies to known proteins; AAF-44,
however, is evolutionarily related to the 32-kDa subunit of replication
protein A (RPA-32) and contains an oligonucleotide/oligosaccharide-binding
(OB) fold domain similar to the OB fold domains of RPA involved in
single-stranded DNA binding. Epitope-tagged versions of AAF-44 and -132 formed
a complex in intact cells, and purified recombinant AAF-44 bound to
single-stranded DNA and stimulated DNA primase activity only in the presence
of AAF-132. Mutations in conserved residues within the OB fold of AAF-44
reduced DNA binding activity of the AAF-44·AAF-132 complex.
Immunofluorescence staining of AAF-44 and AAF-132 in S phase-enriched HeLa
cells demonstrated punctate nuclear staining, and AAF co-localized with
proliferating cell nuclear antigen, a marker for replication foci containing
DNA polymerase-α·primase and RPA. Small interfering RNA-mediated
depletion of AAF-44 in tumor cell lines inhibited
[methyl-3H]thymidine uptake into DNA but did not affect
cell viability. We conclude that AAF shares structural and functional
similarities with RPA-32 and regulates DNA replication, consistent with its
ability to increase polymerase-α·primase template affinity and
stimulate both DNA primase and polymerase-α activities in
vitro.In eukaryotic cells, DNA replication is initiated at multiple origins
internal to each chromosome; the origin recognition complex recruits cell
division cycle and minichromosome maintenance proteins to form a preinitiation
complex (1). At the
G1-S phase transition, the latter complex is activated by
cyclin-dependent protein kinases leading to formation of an initiation complex
that alters local DNA structure through DNA helicase activity
(1,
2). The replication protein A
(RPA)2 is recruited to
bind and stabilize single-stranded DNA (ssDNA) produced by the initiation
complex (3,
4). RPA serves as an auxiliary
factor for DNA polymerase-α (pol-α)·primase: it stabilizes
the protein complex by direct interaction with both pol-α and primase
subunits, and it reduces the misincorporation rate of pol-α, acting as a
“fidelity clamp”
(5,
6). The
pol-α·primase complex consists of four subunits, including the
catalytic pol-α subunit (p185), a regulatory B subunit (p70), and two
primase subunits (p49 and p58). On an ssDNA template, the primase synthesizes
short RNA primers from ribonucleoside triphosphates (rNTPs), which are
elongated by pol-α in the presence of deoxyribonucleoside triphosphates
(dNTPs) to form short DNA fragments. Through mechanisms requiring other
replication factors, pol-α·primase is replaced by the more
processive DNA polymerases pol-δ and pol-ε
(7). Pol-ε synthesizes the
leading strand, whereas pol-δ completes each Okazaki fragment initiated
by pol-α·primase on the lagging strand and proofreads errors made
by pol-α (7). The
initiator RNA and DNA fragments are later removed by nucleases, and the
Okazaki fragments are sealed by DNA ligase
(7).The pol-α·primase complex is the only eukaryotic DNA
polymerase able to initiate DNA synthesis de novo. In addition to
initiating DNA replication and synthesizing Okazaki fragments, it appears to
be one of the final targets of cell cycle checkpoint pathways that couple DNA
replication to DNA damage response
(2,
8). The role of RPA in
initiation, elongation, and completion of lagging strand DNA synthesis has
been thoroughly investigated
(3,
9), but in vitro
studies suggest that some additional factors that promote the rapidity of DNA
replication in vivo are still lacking
(2).In the course of purifying pol-α·primase from extracts of
cultured mouse L1210 cells, we identified a factor we named α-accessory
factor (AAF) that stimulates pol-α·primase activity in
vitro (10,
11). The protein has a native
molecular mass of ∼150 kDa as determined from its sedimentation
coefficient and Stokes radius and is composed of two subunits of ∼132 and
∼44 kDa. AAF stimulates pol-α·primase activity with several
different templates and types of reactions: (i) It stimulates selfprimed
reactions with poly(dT), poly(dI·dT), or single-stranded circular DNA;
(ii) it stimulates primed reactions with poly(dA)·oligo(dT) and
multiply primed DNA in the absence of rNTPs, indicating that it affects
pol-α activity when no primers are being made; and (iii) it stimulates
primase activity on ssDNA in the absence of dNTPs, showing that it can enhance
RNA primer synthesis in the absence of DNA synthesis
(11). AAF increases the
template affinity and processivity of pol-α·primase
(12). AAF is highly specific
for pol-α·primase and has no effect on the other mammalian DNA
polymerases β, γ, or δ or on the DNA
polymerase·primase complexes from Drosophila and
Saccharomyces cerevisiae
(11).The cloning of both AAF subunits based on peptide sequences obtained from
the purified protein allowed us now to further characterize the
AAF-44·AAF-132 complex structurally and functionally. Based on siRNA
experiments in cancer cell lines, AAF appears to regulate DNA replication
in vivo. 相似文献
3.
Negoro S Shibata N Tanaka Y Yasuhira K Shibata H Hashimoto H Lee YH Oshima S Santa R Oshima S Mochiji K Goto Y Ikegami T Nagai K Kato D Takeo M Higuchi Y 《The Journal of biological chemistry》2012,287(7):5079-5090
We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αββα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of Tm = 52 °C, enhanced the protein thermostability by 36 °C (Tm = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000–25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500–3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed. 相似文献
4.
Michael Ogundare Spyridon Theofilopoulos Andrew Lockhart Leslie J. Hall Ernest Arenas Jan Sj?vall A. Gareth Brenton Yuqin Wang William J. Griffiths 《The Journal of biological chemistry》2010,285(7):4666-4679
In this study we have profiled the free sterol content of cerebrospinal fluid by a combination of charge tagging and liquid chromatography-tandem mass spectrometry. Surprisingly, the most abundant cholesterol metabolites were found to be C27 and C24 intermediates of the bile acid biosynthetic pathways with structures corresponding to 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7.170 ± 2.826 ng/ml, mean ± S.D., six subjects), 3β-hydroxycholest-5-en-26-oic acid (0.416 ± 0.193 ng/ml), 7α,x-dihydroxy-3-oxocholest-4-en-26-oic acid (1.330 ± 0.543 ng/ml), and 7α-hydroxy-3-oxochol-4-en-24-oic acid (0.172 ± 0.085 ng/ml), and the C26 sterol 7α-hydroxy-26-norcholest-4-ene-3,x-dione (0.204 ± 0.083 ng/ml), where x is an oxygen atom either on the CD rings or more likely on the C-17 side chain. The ability of intermediates of the bile acid biosynthetic pathways to activate the liver X receptors (LXRs) and the farnesoid X receptor was also evaluated. The acidic cholesterol metabolites 3β-hydroxycholest-5-en-26-oic acid and 3β,7α-dihydroxycholest-5-en-26-oic acid were found to activate LXR in a luciferase assay, but the major metabolite identified in this study, i.e. 7α-hydroxy-3-oxocholest-4-en-26-oic acid, was not an LXR ligand. 7α-Hydroxy-3-oxocholest-4-en-26-oic acid is formed from 3β,7α-dihydroxycholest-5-en-26-oic acid in a reaction catalyzed by 3β-hydroxy-Δ5-C27-steroid dehydrogenase (HSD3B7), which may thus represent a deactivation pathway of LXR ligands in brain. Significantly, LXR activation has been found to reduce the symptoms of Alzheimer disease (Fan, J., Donkin, J., and Wellington C. (2009) Biofactors 35, 239–248); thus, cholesterol metabolites may play an important role in the etiology of Alzheimer disease. 相似文献
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6.
7.
Jennifer A. Thompson Christopher O. Paschall Mike O'Donnell Linda B. Bloom 《The Journal of biological chemistry》2009,284(46):32147-32157
In Escherichia coli, the γ complex clamp loader loads the β-sliding clamp onto DNA. The β clamp tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, γ complex binds β and DNA to form a ternary complex. Binding to primed template DNA triggers γ complex to hydrolyze ATP and release the clamp onto DNA. Here, we investigated the kinetics of forming a ternary complex by measuring rates of γ complex binding β and DNA. A fluorescence intensity-based β binding assay was developed in which the fluorescence of pyrene covalently attached to β increases when bound by γ complex. Using this assay, an association rate constant of 2.3 × 107 m−1 s−1 for γ complex binding β was determined. The rate of β binding was the same in experiments in which γ complex was preincubated with ATP before adding β or added directly to β and ATP. In contrast, when γ complex is preincubated with ATP, DNA binding is faster than when γ complex is added to DNA and ATP at the same time. Slow DNA binding in the absence of ATP preincubation is the result of a rate-limiting ATP-induced conformational change. Our results strongly suggest that the ATP-induced conformational changes that promote β binding and DNA binding differ. The slow ATP-induced conformational change that precedes DNA binding may provide a kinetic preference for γ complex to bind β before DNA during the clamp loading reaction cycle. 相似文献
8.
Armando Durazo Bryan F. Shaw Madhuri Chattopadhyay Kym F. Faull Aram M. Nersissian Joan Selverstone Valentine Julian P. Whitelegge 《The Journal of biological chemistry》2009,284(49):34382-34389
The structure and unfolding of metal-free (apo) human wild-type SOD1 and three pathogenic variants of SOD1 (A4V, G93R, and H48Q) that cause familial amyotrophic lateral sclerosis have been studied with amide hydrogen/deuterium exchange and mass spectrometry. The results indicate that a significant proportion of each of these proteins exists in solution in a conformation in which some strands of the β-barrel (i.e. β2) are well protected from exchange at physiological temperature (37 °C), whereas other strands (i.e. β3 and β4) appear to be unprotected from hydrogen/deuterium exchange. Moreover, the thermal unfolding of these proteins does not result in the uniform incorporation of deuterium throughout the polypeptide but involves the local unfolding of different residues at different temperatures. Some regions of the proteins (i.e. the “Greek key” loop, residues 104–116) unfold at a significantly higher temperature than other regions (i.e. β3 and β4, residues 21–53). Together, these results show that human wild-type apo-SOD1 and variants have a partially unfolded β-barrel at physiological temperature and unfold non-cooperatively. 相似文献
9.
Cecilia Y. Cheng Jie Yang Susan S. Taylor Donald K. Blumenthal 《The Journal of biological chemistry》2009,284(51):35916-35925
The catalytic (C) and regulatory (R) subunits of protein kinase A are exceptionally dynamic proteins. Interactions between the R- and C-subunits are regulated by cAMP binding to the two cyclic nucleotide-binding domains in the R-subunit. Mammalian cells express four different isoforms of the R-subunit (RIα, RIβ, RIIα, and RIIβ) that all interact with the C-subunit in different ways. Here, we investigate the dynamic behavior of protein complexes between RIα and C-subunits using small angle x-ray scattering. We show that a single point mutation in RIα, R333K (which alters the cAMP-binding properties of Domain B) results in a compact shape compared with the extended shape of the wild-type R·C complex. A double mutant complex that disrupts the interaction site between the C-subunit and Domain B in RIα, RIαABR333K·C(K285P), results in a broader P(r) curve that more closely resembles the P(r) profiles of wild-type complexes. These results together suggest that interactions between RIα Domain B and the C-subunit in the RIα·C complex involve large scale dynamics that can be disrupted by single point mutations in both proteins. In contrast to RIα·C complexes. Domain B in the RIIβ·C heterodimer is not dynamic and is critical for both inhibition and complex formation. Our study highlights the functional differences of domain dynamics between protein kinase A isoforms, providing a framework for elucidating the global organization of each holoenzyme and the cross-talk between the R- and C-subunits. 相似文献
10.
Proteins in aggregates functionally impact multiple neurodegenerative disease models by forming proteasome‐blocking complexes
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Age-dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP-43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation-prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc-54p/Q40::YFP, a model of polyglutamine array diseases such as Huntington''s, accrues aggregates in muscle 2–6 days posthatch. These foci, isolated on antibody-coupled magnetic beads, were characterized by high-resolution mass spectrometry. Three Q40::YFP-associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimer''s disease models, nematodes with muscle or pan-neuronal Aβ1–42 expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine-rich, favoring hydrophobic interactions with other random-coil domains. A particularly potent modulator of aggregation, CRAM-1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72–86% (P < 10−6). In worms expressing Aβ1–42, knockdown of cram-1 reduced β-amyloid 60% (P < 0.002) and slowed age-dependent paralysis > 30% (P < 10−6). In wild-type worms, cram-1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM-1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM-1 (or human orthologs) with ubiquitin, and we verified that CRAM-1 reduces degradation of a tagged-ubiquitin reporter. We propose that CRAM-1 exemplifies a class of primitive chaperones that are initially protective and highly beneficial for early reproduction, but ultimately impair aggregate clearance and limit longevity. 相似文献
11.
Ronggai Li 《Cytotechnology》2015,67(6):987-993
A practical method was developed for the transient transfection of Chinese hamster ovary (CHO) cells with 25 kDa linear polyethylenimine (PEI) then optimal culture conditions determined for the production of rainbow trout (Oncorhynchus mykiss) IFN-γ recombinant protein. We found that culture temperature had a significant impact upon recombinant protein yield, with best results being obtained at 32 °C. However the amount of serum added to the culture medium had no effect upon recombinant IFN-γ (rIFN-γ) production. In this study maximal rIFN-γ yields and minimal PEI toxicity were achieved using a DNA/PEI ratio of 1:8, where the amount of PEI did not exceed 10 µg per 5 ml of RPMI1640 culture medium, with cells subsequently cultured at 32 °C for 7 days. Thus, linear PEI is a technically simple and cost-efficient method for the transient transfection of CHO cells and is compatible with serum-free operations. 相似文献
12.
Michael Helwig Akina Hoshino Casey Berridge Sang-Nam Lee Nikolai Lorenzen Daniel E. Otzen Jason L. Eriksen Iris Lindberg 《The Journal of biological chemistry》2013,288(2):1114-1124
Neurodegenerative diseases such as Alzheimer (AD) and Parkinson (PD) are characterized by abnormal aggregation of misfolded β-sheet-rich proteins, including amyloid-β (Aβ)-derived peptides and tau in AD and α-synuclein in PD. Correct folding and assembly of these proteins are controlled by ubiquitously expressed molecular chaperones; however, our understanding of neuron-specific chaperones and their involvement in the pathogenesis of neurodegenerative diseases is limited. We here describe novel chaperone-like functions for the secretory protein 7B2, which is widely expressed in neuronal and endocrine tissues. In in vitro experiments, 7B2 efficiently prevented fibrillation and formation of Aβ1–42, Aβ1–40, and α-synuclein aggregates at a molar ratio of 1:10. In cell culture experiments, inclusion of recombinant 7B2, either in the medium of Neuro-2A cells or intracellularly via adenoviral 7B2 overexpression, blocked the neurocytotoxic effect of Aβ1–42 and significantly increased cell viability. Conversely, knockdown of 7B2 by RNAi increased Aβ1–42-induced cytotoxicity. In the brains of APP/PSEN1 mice, a model of AD amyloidosis, immunoreactive 7B2 co-localized with aggregation-prone proteins and their respective aggregates. Furthermore, in the hippocampus and substantia nigra of human AD- and PD-affected brains, 7B2 was highly co-localized with Aβ plaques and α-synuclein deposits, strongly suggesting physiological association. Our data provide insight into novel functions of 7B2 and establish this neural protein as an anti-aggregation chaperone associated with neurodegenerative disease. 相似文献
13.
Fungal keratitis is a serious corneal disease that may result in loss of vision. There are limited treatment options available in Iraqi eye hospitals which might be the main reason behind the poor prognosis of many cases. The purpose of this study was to prepare and pharmaceutically evaluate clotrimazole–β-cyclodextrin (CTZ–β-CD) eyedrops then clinically assess its therapeutic efficacy on fungal keratitis compared with extemporaneous amphotericin B eyedrops (0.5% w/v). A CTZ–β-CD ophthalmic solution was prepared and evaluated by various physicochemical, microbiological, and biological tests. The prepared formula was stable in 0.05 M phosphate buffer pH 7.0 at 40 ± 2°C and 75 ± 5% RH for a period of 6 months. Light has no significant effect on the formula’s stability. The CTZ–β-CD eyedrops efficiently complied with the isotonicity, sterility, and antimicrobiological preservative effectiveness tests. Results of the clinical study revealed that 20 (80%) patients showed a favorable response to the CTZ–β-CD eyedrops, while 16 patients (64%) exhibited a favorable response to amphotericin B (P > 0.05). The mean course of treatment was significantly (P < 0.05) less in the CTZ treatment group than in the amphotericin group (21.5 ± 5.2 vs. 28.3 ± 6.4 days, respectively). The CTZ formulation was significantly (P < 0.05) more effective in the management of severe cases and also against Candida sp. than amphotericin B. There was no significant difference (P < 0.05) between both therapies against filamentous fungi. The CTZ–β-CD formulation can be used alternatively to other ophthalmic antimycotic treatment options in developing countries where stability, cost, or efficacy is a limiting factor.Key words: clotrimazole, β-cyclodextrin, eyedrops, fungal keratitis, Iraq 相似文献
14.
Amandine Gastebois Isabelle Mouyna Catherine Simenel C��cile Clavaud Bernadette Coddeville Muriel Delepierre Jean-Paul Latg�� Thierry Fontaine 《The Journal of biological chemistry》2010,285(4):2386-2396
A new HPLC method was developed to separate linear from β(1–6)-branched β(1–3)-glucooligosaccharides. This methodology has permitted the isolation of the first fungal β(1–6)/β(1–3)-glucan branching transglycosidase using a cell wall autolysate of Aspergillus fumigatus (Af). The encoding gene, AfBGT2 is an ortholog of AfBGT1, another transglycosidase of A. fumigatus previously analyzed (Mouyna, I., Hartland, R. P., Fontaine, T., Diaquin, M., Simenel, C., Delepierre, M., Henrissat, B., and Latgé, J. P. (1998) Microbiology 144, 3171–3180). Both enzymes release laminaribiose from the reducing end of a β(1–3)-linked oligosaccharide and transfer the remaining chain to another molecule of the original substrate. The AfBgt1p transfer occurs at C-6 of the non-reducing end group of the acceptor, creating a kinked β(1–3;1–6) linear molecule. The AfBgt2p transfer takes place at the C-6 of an internal group of the acceptor, resulting in a β(1–3)-linked product with a β(1–6)-linked side branch. The single Afbgt2 mutant and the double Afbgt1/Afbgt2 mutant in A. fumigatus did not display any cell wall phenotype showing that these activities were not responsible for the construction of the branched β(1–3)-glucans of the cell wall. 相似文献
15.
Zinc (Zn) is an essential component of Zn-finger proteins and acts as a cofactor for enzymes required for cellular metabolism and in the maintenance of DNA integrity. The study investigated the genotoxic and cytotoxic effects of Zn deficiency or excess in a primary human oral keratinocyte cell line and determined the optimal concentration of two Zn compounds (Zn Sulphate (ZnSO4) and Zn Carnosine (ZnC)) to minimise DNA damage. Zn-deficient medium (0 μM) was produced using Chelex treatment, and the two Zn compounds ZnSO4 and ZnC were tested at concentrations of 0.0, 0.4, 4.0, 16.0, 32.0 and 100.0 μM. Cell viability was decreased in Zn-depleted cells (0 μM) as well as at 32 μM and 100 μM for both Zn compounds (P < 0.0001) as measured via the MTT assay. DNA strand breaks, as measured by the comet assay, were found to be increased in Zn-depleted cells compared with the other treatment groups (P < 0.05). The Cytokinesis Block Micronucleus Cytome assay showed a significant increase in the frequency of both apoptotic and necrotic cells under Zn-deficient conditions (P < 0.05). Furthermore, elevated frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBuds) were observed at 0 and 0.4 μM Zn, whereas these biomarkers were minimised for both Zn compounds at 4 and 16 μM Zn (P < 0.05), suggesting these concentrations are optimal to maintain genome stability. Expression of PARP, p53 and OGG1 measured by western blotting was increased in Zn-depleted cells indicating that DNA repair mechanisms are activated. These results suggest that maintaining Zn concentrations within the range of 4–16 μM is essential for DNA damage prevention in cultured human oral keratinocytes. 相似文献
16.
We report a systematic study of the condensation of plasmid DNA by oligocations with variation of the charge, Z, from +3 to +31. The oligocations include a series of synthetic linear ε-oligo(l-lysines), (denoted εKn, n = 3–10, 31; n is the number of lysines with the ligand charge Z = n+1) and branched α-substituted homologues of εK10: εYK10, εLK10 (Z = +11); εRK10, εYRK10 and εLYRK10 (Z = +21). Data were obtained by light scattering, UV absorption monitored precipitation assay and isothermal titration calorimetry in a wide range concentrations of DNA and monovalent salt (KCl, CKCl). The dependence of EC50 (ligand concentration at the midpoint of DNA condensation) on CKCl shows the existence of a salt-independent regime at low CKCl and a salt-dependent regime with a steep rise of EC50 with increase of CKCl. Increase of the ligand charge shifts the transition from the salt-independent to salt-dependent regime to higher CKCl. A novel and simple relationship describing the EC50 dependence on DNA concentration, charge of the ligand and the salt-dependent dissociation constant of the ligand–DNA complex is derived. For the ε-oligolysines εK6–εK10, the experimental dependencies of EC50 on CKCl and Z are well-described by an equation with a common set of parameters. Implications from our findings for understanding DNA condensation in chromatin are discussed. 相似文献
17.
TI Pollin T Isakova KA Jablonski PI de Bakker A Taylor J McAteer Q Pan ES Horton LM Delahanty D Altshuler AR Shuldiner RB Goldberg JC Florez PW Franks;Diabetes Prevention Program Research Group 《PLoS genetics》2012,8(8):e1002895
Weight-loss interventions generally improve lipid profiles and reduce cardiovascular disease risk, but effects are variable and may depend on genetic factors. We performed a genetic association analysis of data from 2,993 participants in the Diabetes Prevention Program to test the hypotheses that a genetic risk score (GRS) based on deleterious alleles at 32 lipid-associated single-nucleotide polymorphisms modifies the effects of lifestyle and/or metformin interventions on lipid levels and nuclear magnetic resonance (NMR) lipoprotein subfraction size and number. Twenty-three loci previously associated with fasting LDL-C, HDL-C, or triglycerides replicated (P = 0.04–1×10−17). Except for total HDL particles (r = −0.03, P = 0.26), all components of the lipid profile correlated with the GRS (partial |r| = 0.07–0.17, P = 5×10−5–1×10−19). The GRS was associated with higher baseline-adjusted 1-year LDL cholesterol levels (β = +0.87, SEE±0.22 mg/dl/allele, P = 8×10−5, P
interaction = 0.02) in the lifestyle intervention group, but not in the placebo (β = +0.20, SEE±0.22 mg/dl/allele, P = 0.35) or metformin (β = −0.03, SEE±0.22 mg/dl/allele, P = 0.90; P
interaction = 0.64) groups. Similarly, a higher GRS predicted a greater number of baseline-adjusted small LDL particles at 1 year in the lifestyle intervention arm (β = +0.30, SEE±0.012 ln nmol/L/allele, P = 0.01, P
interaction = 0.01) but not in the placebo (β = −0.002, SEE±0.008 ln nmol/L/allele, P = 0.74) or metformin (β = +0.013, SEE±0.008 nmol/L/allele, P = 0.12; P
interaction = 0.24) groups. Our findings suggest that a high genetic burden confers an adverse lipid profile and predicts attenuated response in LDL-C levels and small LDL particle number to dietary and physical activity interventions aimed at weight loss. 相似文献
18.
The energy functions used to predict protein structures typically include both molecular-mechanics and knowledge-based terms. In contrast, our approach is to develop robust physics- and geometry-based methods. Here, we investigate to what extent simple hard-sphere models can be used to predict side-chain conformations. The distributions of the side-chain dihedral angle χ1 of Val and Thr in proteins of known structure show distinctive features: Val side chains predominantly adopt χ1 = 180°, whereas Thr side chains typically adopt χ1 = 60° and 300° (i.e., χ1 = ±60° or g− and g+ configurations). Several hypotheses have been proposed to explain these differences, including interresidue steric clashes and hydrogen-bonding interactions. In contrast, we show that the observed side-chain dihedral angle distributions for both Val and Thr can be explained using only local steric interactions in a dipeptide mimetic. Our results emphasize the power of simple physical approaches and their importance for future advances in protein engineering and design. 相似文献
19.
M?ssbauer spectroscopy of the nitrogenase proteins from Klebsiella pneumoniae. Structural assignments and mechanistic conclusions 总被引:14,自引:12,他引:2
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The Mo–Fe protein and the Fe protein which together constitute the nitrogenase of Klebsiella pneumoniae were prepared from bacteria grown in 57Fe-enriched medium. The Mössbauer spectrum of the Mo–Fe protein, as isolated in the presence of Na2S2O4, showed that the protein contained three iron species, called M4, M5 and M6. The area of the spectrum associated with species M4, with δ=0.65mm/s and ΔE=3.05mm/s at 4.2°K, corresponded to two iron atoms/molecule of protein and it is interpreted as being due to a high-spin ferrous, spin-coupled pair of iron atoms. The iron atoms of species M4 may be involved in the quaternary structure of the protein. Species M5, with δ=0.61mm/s and ΔE=0.83mm/s at 77°K, corresponded to eight iron atoms/molecule of protein and is interpreted as being due to Fe4S4 or Fe2S2 low-spin ferrous iron clusters. Species M6, with δ=0.37mm/s and ΔE=0.71mm/s at 77°K, also corresponded to eight iron atoms/molecule of protein and, at 4.2°K, became a broad shallow absorption, characteristic of magnetic hyperfine interaction. Oxidation of the Mo–Fe protein with the redox dye Lauth''s Violet did not affect the activity of the protein but changed species M4, M5 and M6 into the species M1 (δ=0.37mm/s, ΔE=0.75mm/s at 77°K, broad magnetic component at 4.2°K) and M2 (δ=0.35mm/s, ΔE=0.9mm/s at 4.2°K). In the presence of the Fe protein, Na2S2O4, ATP and Mg2+, the M6 component of the Mo–Fe protein was replaced by species M7 with δ=0.46mm/s, ΔE=1.04mm/s at 4.2°K. The change in Mössbauer parameters associated with the M6 → M7 transformation was very similar to the change observed on reduction of the high-potential Fe protein from Chromatium vinosum. In contrast, Na2S2O4-reduced Fe protein contained only one type of iron cluster (F4). Species F4 had δ=0.50mm/s, ΔE=0.9mm/s at 195°K, and at 4.2°K broadened in a manner characteristic of a magnetic hyperfine interaction, associated with half-integral spin, equally distributed over all four atoms of the Fe protein. The Mössbauer spectra of the Mo–Fe and the Fe protein under argon were unaffected by the reducible substrates N2 and C2H2 and the inhibitor CO in the presence of ATP, Mg2+ and Na2S2O4. A number of Mössbauer spectral species associated with inactivated Mo–Fe and Fe proteins are described and discussed. 相似文献
20.
Ikuo Masuho Jeremy Celver Abraham Kovoor Kirill A. Martemyanov 《The Journal of biological chemistry》2010,285(7):4781-4787
The R7 subfamily of RGS proteins critically regulates neuronal G protein-signaling pathways that are essential for vision, nociception, motor coordination, and reward processing. A member of the R7 RGS family, RGS11, is a GTPase-accelerating protein specifically expressed in retinal ON-bipolar cells where it forms complexes with the atypical G protein β subunit, Gβ5, and transmembrane protein R9AP. Association with R9AP has been shown to be critical for the proteolytic stability of the complex in the retina. In this study we report that R9AP can in addition stimulate the GTPase-accelerating protein activity of the RGS11·Gβ5 complex at Gαo. Single turnover GTPase assays reveal that R9AP co-localizes RGS11·Gβ5 and Gαo on the membrane and allosterically potentiates the GTPase-accelerating function of RGS11·Gβ5. Reconstitution of mGluR6-Gαo signaling in Xenopus oocytes indicates that RGS11·Gβ5-mediated GTPase acceleration in this system requires co-expression of R9AP. The results provide new insight into the regulation of mGluR6-Gαo signaling by the RGS11·Gβ5·R9AP complex and establish R9AP as a general GTPase-accelerating protein activity regulator of R7 RGS complexes. 相似文献