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51.
Sabbattini P Canzonetta C Sjoberg M Nikic S Georgiou A Kemball-Cook G Auner HW Dillon N 《The EMBO journal》2007,26(22):4657-4669
Combinatorial modifications of the core histones have the potential to fine-tune the epigenetic regulation of chromatin states. The Aurora B kinase is responsible for generating the double histone H3 modification tri-methylated K9/phosphorylated S10 (H3K9me3/S10ph), which has been implicated in chromosome condensation during mitosis. In this study, we have identified a novel role for Aurora B in epigenetic marking of silent chromatin during cell differentiation. We find that phosphorylation of H3 S10 by Aurora B generates high levels of the double H3K9me3/S10ph modification in differentiated postmitotic cells and also results in delocalisation of HP1beta away from heterochromatin in terminally differentiated plasma cells. Microarray analysis of the H3K9me3/S10ph modification shows a striking increase in the modification across repressed genes during differentiation of mesenchymal stem cells. Our results provide evidence that the Aurora B kinase has a role in marking silent chromatin independently of the cell cycle and suggest that targeting of Aurora B-mediated phosphorylation of H3 S10 to repressed genes could be a mechanism for epigenetic silencing of gene expression. 相似文献
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Vasiliki Daraki Vaggelis Georgiou Stathis Papavasiliou Georgia Chalkiadaki Marianna Karahaliou Stella Koinaki Katerina Sarri Maria Vassilaki Manolis Kogevinas Leda Chatzi 《PloS one》2015,10(5)
ContextMaternal pre-pregnancy obesity may increase the risk of childhood obesity but it is unknown whether other metabolic factors in early pregnancy such as lipid profile and hypertension are associated with offspring cardiometabolic traits.ObjectiveOur objective was to investigate whether fasting lipid, glucose, and insulin levels during early pregnancy and maternal pre-pregnancy weight status, are associated with offspring adiposity measures, lipid levels and blood pressure at preschool age.ResultsPre-pregnancy overweight/obesity was associated with greater risk of offspring overweight/obesity (RR: 1.83, 95%CI: 1.19, 2.81), central adiposity (RR: 1.97, 95%CI: 1.11, 3.49), and greater fat mass by 5.10mm (95%CI: 2.49, 7.71) at 4 years of age. These associations were more pronounced in girls. An increase of 40mg/dl in fasting serum cholesterol levels in early pregnancy was associated with greater skinfold thickness by 3.30mm (95%CI: 1.41, 5.20) at 4 years of age after adjusting for pre-pregnancy BMI and several other confounders. An increase of 10mmHg in diastolic blood pressure in early pregnancy was associated with increased risk of offspring overweight/obesity (RR: 1.22, 95%CI: 1.03, 1.45), and greater skinfold thickness by 1.71mm (95% CI: 0.57, 2.86) at 4 years of age.ConclusionsMetabolic dysregulation in early pregnancy may increase the risk of obesity at preschool age. 相似文献
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Theta class glutathione transferases (GST) from various species exhibit markedly different catalytic activities in conjugating the tripeptide glutathione (GSH) to a variety of electrophilic substrates. For example, the human theta 1-1 enzyme (hGSTT1-1) is 440-fold less efficient than the rat theta 2-2 enzyme (rGSTT2-2) with the fluorogenic substrate 7-amino-4-chloromethyl coumarin (CMAC). Large libraries of hGSTT1-1 constructed by error-prone PCR, DNA shuffling, or saturation mutagenesis were screened for improved catalytic activity towards CMAC in a quantitative fashion using flow cytometry. An iterative directed evolution approach employing random mutagenesis in conjunction with homologous recombination gave rise to enzymes exhibiting up to a 20,000-fold increase in k(cat)/K(M) compared to hGSTT1-1. All highly active clones encoded one or more mutations at residues 32, 176, or 234. Combinatorial saturation mutagenesis was used to evaluate the full complement of natural amino acids at these positions, and resulted in the isolation of enzymes with catalytic rates comparable to those exhibited by the fastest mutants obtained via directed evolution. The substrate selectivities of enzymes resulting from random mutagenesis, DNA shuffling, and combinatorial saturation mutagenesis were evaluated using a series of distinct electrophiles. The results revealed that promiscuous substrate activities arose in a stochastic manner, as they did not correlate with catalytic efficiency towards the CMAC selection substrate. In contrast, chimeric enzymes previously constructed by homology-independent recombination of hGSTT-1 and rGSTT2-2 exhibited very different substrate promiscuity profiles, and showed a more defined relationship between evolved and promiscuous activities. 相似文献
55.
The expression of heterologous secreted proteins in Escherichia coli is widely employed for laboratory and preparative purposes. Thanks to advances in expression technologies over the past 25 years, many mammalian proteins can now be produced routinely in secreted form with yields in the gram/litre scale. Nonetheless, ensuring efficient secretion across the inner membrane, and preventing proteolytic degradation, incorrect disulfide-bond formation and aggregation into periplasmic inclusion bodies, frequently presents significant challenges. Recent advances in the understanding of the periplasmic folding quality control system are leading to new strategies to facilitate the expression of heterologous secreted proteins. In parallel, protein design and directed evolution approaches are beginning to be exploited for engineering of the cellular protein folding machinery to achieve further improvements in protein expression. 相似文献
56.
Abnormal fractionation of beta-lactamase in Escherichia coli: evidence for an interaction with the inner membrane in the absence of a leader peptide.
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beta-Lactamase with the -20 to -1 region of the leader peptide deleted (almost complete deletion of the leader peptide) [delta(-20,-1) beta-lactamase] was released from Escherichia coli cells by osmotic shock. Fractionation of the cells by conversion to spheroplasts and protease accessibility experiments further indicated that a portion of the protein may be bound to the cytoplasmic membrane and be partially exposed in the periplasmic space. Expression of delta(-20,-1) beta-lactamase conferred a 25-fold increase in the 50% lethal dose for ampicillin relative to that for controls, thus confirming that a small amount (about 2%) of the active protein is completely exported from the cytoplasm. These results suggest that even in the absence of a leader peptide, mature beta-lactamase is able to interact with the cytoplasmic membrane and be translocated into the periplasmic space, albeit with a low efficiency. 相似文献
57.
Efthymios Maneas Rosalind Aughwane Nam Huynh Wenfeng Xia Rehman Ansari Mithun Kuniyil Ajith Singh J. Ciaran Hutchinson Neil J. Sebire Owen J. Arthurs Jan Deprest Sebastien Ourselin Paul C. Beard Andrew Melbourne Tom Vercauteren Anna L. David Adrien E. Desjardins 《Journal of biophotonics》2020,13(4)
Minimally invasive fetal interventions require accurate imaging from inside the uterine cavity. Twin‐to‐twin transfusion syndrome (TTTS), a condition considered in this study, occurs from abnormal vascular anastomoses in the placenta that allow blood to flow unevenly between the fetuses. Currently, TTTS is treated fetoscopically by identifying the anastomosing vessels, and then performing laser photocoagulation. However, white light fetoscopy provides limited visibility of placental vasculature, which can lead to missed anastomoses or incomplete photocoagulation. Photoacoustic (PA) imaging is an alternative imaging method that provides contrast for hemoglobin, and in this study, two PA systems were used to visualize chorionic (fetal) superficial and subsurface vasculature in human placentas. The first system comprised an optical parametric oscillator for PA excitation and a 2D Fabry‐Pérot cavity ultrasound sensor; the second, light emitting diode arrays and a 1D clinical linear‐array ultrasound imaging probe. Volumetric photoacoustic images were acquired from ex vivo normal term and TTTS‐treated placentas. It was shown that superficial and subsurface branching blood vessels could be visualized to depths of approximately 7 mm, and that ablated tissue yielded negative image contrast. This study demonstrated the strong potential of PA imaging to guide minimally invasive fetal therapies. 相似文献
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D. Brent Halling Dimitra K. Georgiou D. J. Black Guojun Yang Jennifer L. Fallon Florante A. Quiocho Steen E. Pedersen Susan L. Hamilton 《The Journal of biological chemistry》2009,284(30):20041-20051
Calmodulin binds to IQ motifs in the α1 subunit of CaV1.1 and CaV1.2, but the affinities of calmodulin for the motif and for Ca2+ are higher when bound to CaV1.2 IQ. The CaV1.1 IQ and CaV1.2 IQ sequences differ by four amino acids. We determined the structure of calmodulin bound to CaV1.1 IQ and compared it with that of calmodulin bound to CaV1.2 IQ. Four methionines in Ca2+-calmodulin form a hydrophobic binding pocket for the peptide, but only one of the four nonconserved amino acids (His-1532 of CaV1.1 and Tyr-1675 of CaV1.2) contacts this calmodulin pocket. However, Tyr-1675 in CaV1.2 contributes only modestly to the higher affinity of this peptide for calmodulin; the other three amino acids in CaV1.2 contribute significantly to the difference in the Ca2+ affinity of the bound calmodulin despite having no direct contact with calmodulin. Those residues appear to allow an interaction with calmodulin with one lobe Ca2+-bound and one lobe Ca2+-free. Our data also provide evidence for lobe-lobe interactions in calmodulin bound to CaV1.2.The complexity of eukaryotic Ca2+ signaling arises from the ability of cells to respond differently to Ca2+ signals that vary in amplitude, duration, and location. A variety of mechanisms decode these signals to drive the appropriate physiological responses. The Ca2+ sensor for many of these physiological responses is the Ca2+-binding protein calmodulin (CaM).2 The primary sequence of CaM is tightly conserved in all eukaryotes, yet it binds and regulates a broad set of target proteins in response to Ca2+ binding. CaM has two domains that bind Ca2+ as follows: an amino-terminal domain (N-lobe) and a carboxyl-terminal domain (C-lobe) joined via a flexible α-helix. Each lobe of CaM binds two Ca2+ ions, and binding within each lobe is highly cooperative. The two lobes of CaM, however, have distinct Ca2+ binding properties; the C-lobe has higher Ca2+ affinity because of a slower rate of dissociation, whereas the N-lobe has weaker Ca2+ affinity and faster kinetics (1). CaM can also bind to some target proteins in both the presence and absence of Ca2+, and the preassociation of CaM in low Ca2+ modulates the apparent Ca2+ affinity of both the amino-terminal and carboxyl-terminal lobes. Differences in the Ca2+ binding properties of the lobes and in the interaction sites of the amino- and carboxyl-terminal lobes enable CaM to decode local versus global Ca2+ signals (2).Even though CaM is highly conserved, CaM target (or recognition) sites are quite heterogeneous. The ability of CaM to bind to very different targets is at least partially due to its flexibility, which allows it to assume different conformations when bound to different targets. CaM also binds to various targets in distinct Ca2+ saturation states as follows: Ca2+-free (3), Ca2+ bound to only one of the two lobes, or fully Ca2+-bound (4–7). In addition, CaM may bind with both lobes bound to a target (5, 6) or with only a single lobe engaged (8). If a target site can bind multiple conformers of CaM, CaM may undergo several transitions that depend on Ca2+ concentration, thereby tuning the functional response. Identification of stable intermediate states of CaM bound to individual targets will help to elucidate the steps involved in this fine-tuned control.Both CaV1.1 and CaV1.2 belong to the L-type family of voltage-dependent Ca2+ channels, which bind apoCaM and Ca2+-CaM at carboxyl-terminal recognition sites in their α1 subunits (9–14). Ca2+ binding to CaM, bound to CaV1.2 produces Ca2+-dependent facilitation (CDF) (14). Whether CaV1.1 undergoes CDF is not known. However, both CaV1.2 and CaV1.1 undergo Ca2+- and CaM-dependent inactivation (CDI) (14, 15). CaV1.1 CDI is slower and more sensitive to buffering by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid than CaV1.2 CDI (15). Ca2+ buffers are thought to influence CDI and/or CDF in voltage-dependent Ca2+ channels by competing with CaM for Ca2+ (16).The conformation of the carboxyl terminus of the α1 subunit is critical for channel function and has been proposed to regulate the gating machinery of the channel (17, 18). Several interactions of this region include intramolecular contacts with the pore inactivation machinery and intermolecular contacts with CaM kinase II and ryanodine receptors (17, 19–22). Ca2+ regulation of CaV1.2 may involve several motifs within this highly conserved region, including an EF hand motif and three contiguous CaM-binding sequences (10, 12). ApoCaM and Ca2+-CaM-binding sites appear to overlap at the site designated as the “IQ motif” (9, 12, 13), which are critical for channel function at the molecular and cellular level (14, 23).Differences in the rate at which 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid affects CDI of CaV1.1 and CaV1.2 could reflect differences in their interactions with CaM. In this study we describe the differences in CaM interactions with the IQ motifs of the CaV1.1 and the CaV1.2 channels in terms of crystal structure, CaM affinity, and Ca2+ binding to CaM. We find the structures of Ca2+-CaM-IQ complexes are similar except for a single amino acid change in the peptide that contributes to its affinity for CaM. We also find that the other three amino acids that differ in CaV1.2 and CaV1.1 contribute to the ability of CaV1.2 to bind a partially Ca2+-saturated form of CaM. 相似文献