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1.  [No title available] 
PMCID: PMC4048651  PMID: 24314137
2.  Nuclear Raf-1 kinase regulates CXCR5 promoter by associating with NFATc3 to drive retinoic acid-induced leukemic cell differentiation 
The FEBS journal  2014;281(4):1170-1180.
Novel functions of signaling molecules have been revealed in studies of cancer stem cells. Retinoic acid (RA) is an embryonic morphogen and stem cell regulator that controls the differentiation of a patient derived leukemic cell line, HL-60, which is composed of progenitor cells with bipotent myelo-monocytic differentiation capability. RA treatment of HL-60 cells causes unusually long-lasting MAPK signaling, with the cells exhibiting the beginning of G0 cell cycle arrest and functional differentiation by 48 hours after treatment with RA. This event coincides with nuclear translocation of Raf-1, phosphorylated at serine 621. This study shows how the novel localization of Raf-1 to the nucleus effects transcriptional changes that contribute to the differentiation of HL-60 cells induced by RA. We find nuclear pS621 Raf-1 associates with NFATc3 near its cognate binding site in the promoter of CXCR5, a gene that must be up-regulated to drive RA-induced differentiation. NFATc3 becomes immunoprecipitable with an anti-phosphoserine antibody, and CXCR5 is transcriptionally up-regulated upon RA-induced differentiation. Inhibiting the pS621 Raf-1/NFATc3 association with PD98059 inhibits these processes and cripples RA-induced differentiation. In this novel paradigm for Raf-1 and RA function, Raf-1 has a role in driving the nuclear signaling of RA-induced differentiation of leukemic progenitor cells.
PMCID: PMC4057990  PMID: 24330068
differentiation; retinoic acid; Raf-1; NFATc3; CXCR5
3.  [No title available] 
PMCID: PMC3916181  PMID: 24289040
4.  An auto-inhibitory effect of the homothorax domain of Meis2 
The FEBS journal  2010;277(12):2584-2597.
Meis2 is a homeodomain protein containing a conserved homothorax (Hth) domain that is present in all Meis and Prep family proteins and in the Drosophila homothorax protein. The Hth domain mediates interaction with Pbx homeodomain proteins, allowing for efficient DNA binding. Here we show that, like Meis1, Meis2 has a strong carboxyl-terminal transcriptional activation domain, which is required for full activation of transcription by homeodomain protein complexes comprised of Meis2 and Pbx1. We also show that the activity of the activation domain is inhibited by the Hth domain, and that this auto-inhibition can be partially relieved by the interaction of Pbx1 with the Hth domain of Meis2. Targeting the Hth domain to DNA suggests that it is not a portable trans-acting repression domain. However, the Hth domain can inhibit a linked activation domain, and this inhibition is not limited to the Meis2 activation domain. Database searching reveals that the Meis3.2 splice variant, which is found in several vertebrate species, disrupts the Hth domain by removing 17 codons from the 5’ end of exon 6. We show that the equivalent deletion in Meis2 derepresses the carboxyl-terminal activation domain and weakens interaction with Pbx1. This work suggests that the transcriptional activity of all members of the Meis/Prep homothorax protein family is subject to auto-inhibition by their Hth domains, and that the Meis3.2 splice variant encodes a protein which bypasses this auto-inhibitory effect.
PMCID: PMC4306237  PMID: 20553494
Meis; Pbx; homeodomain; transcription; repression
5.  PI3K/Akt signalling-mediated protein surface expression sensed by 14-3-3 interacting motif 
The FEBS journal  2009;276(19):5547-5558.
The regulation of protein expression on the cell surface membrane is an important component of the cellular response to extracellular signalling. The translation of extracellular signalling into specific protein localization often involves the post-translational modification of cargo proteins. Using a genetic screen of random peptides, we have previously identified a group of C-terminal sequences, represented by RGRSWTY-COOH (termed ‘SWTY’), which are capable of overriding an endoplasmic reticulum localization signal and directing membrane proteins to the cell surface via specific binding to 14-3-3 proteins. The identity of the kinase signalling pathways that drive phosphorylation and 14-3-3 binding of the SWTY sequence is not known. In this study, we report that the activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway by the over-expression of active kinases, stimulation with fetal bovine serum or growth factors can: (a) phosphorylate the SWTY sequence; (b) recruit 14-3-3 proteins to SWTY; and (c) promote surface expression of the chimeric potassium channel fused with the SWTY sequence. The expression of the dominant negative Akt inhibited the enhancement of surface expression by fetal bovine serum. In addition, the activation of PI3K significantly enhanced the 14-3-3 association and cell surface expression of GPR15, a G protein-coupled receptor which carries an endogenous SWTY-like, C-terminal, 14-3-3 binding sequence and is known to serve as a HIV co-receptor. Given the wealth and specificity of both kinase activity and 14-3-3 binding sequences, our results suggest that the C-terminal SWTY-like motif may serve as a sensor that can selectively induce the cell surface expression of membrane proteins in response to different extracellular signals.
PMCID: PMC4301307  PMID: 19691494
14-3-3; Akt; GPR15; PI3K; regulation of surface expression
6.  [No title available] 
PMCID: PMC4290664  PMID: 19459942
7.  Crystal structure of the antimicrobial peptidase lysostaphin from Staphylococcus simulans 
The Febs Journal  2014;281(18):4112-4122.
Staphylococcus simulans biovar staphylolyticus lysostaphin efficiently cleaves Staphylococcus aureus cell walls. The protein is in late clinical trials as a topical anti-staphylococcal agent, and can be used to prevent staphylococcal growth on artificial surfaces. Moreover, the gene has been both stably engineered into and virally delivered to mice or livestock to obtain resistance against staphylococci. Here, we report the first crystal structure of mature lysostaphin and two structures of its isolated catalytic domain at 3.5, 1.78 and 1.26 Å resolution, respectively. The structure of the mature active enzyme confirms its expected organization into catalytic and cell-wall-targeting domains. It also indicates that the domains are mobile with respect to each other because of the presence of a highly flexible peptide linker. The high-resolution structures of the catalytic domain provide details of Zn2+ coordination and may serve as a starting point for the engineering of lysostaphin variants with improved biotechnological characteristics.
Structured digital abstract
lysostaphin by x-ray crystallography (1,2).
PMCID: PMC4286107  PMID: 25039253
crystal structure; lysis; lysostaphin; Staphylococcus aureus; Staphylococcus simulans
8.  Harnessing the genome for characterization of GPCRs in cancer pathogenesis 
The FEBS journal  2013;280(19):4729-4738.
G-protein coupled receptors (GPCRs) mediate numerous physiological processes and represent the targets for a vast array of therapeutics for diseases ranging from depression to hypertension to reflux. Despite the recognition that GPCRs can act as oncogenes and tumor suppressors by regulating oncogenic signaling networks, few drugs targeting GPCRs are utilized in cancer therapy. Recent large-scale genome-wide analyses of multiple human tumors have uncovered novel GPCRs altered in cancer. However, the work of determining which GPCRs from these lists are drivers of tumorigenesis, and hence valid therapeutic targets, remains a formidable challenge. In this review I will highlight recent studies providing evidence that GPCRs are relevant targets for cancer therapy through their effects on known cancer signaling pathways, tumor progression, invasion and metastasis, and the microenvironment. Furthermore, I will explore how genomic analysis is beginning to shine a light on GPCRs as therapeutic targets in the age of personalized medicine.
PMCID: PMC4283816  PMID: 23927072
GPCR; cancer; bioinformatics; signal transduction; microenvironment; metastasis; genomics
9.  Improved asymmetry prediction for siRNAs 
The FEBS journal  2014;281(1):320-330.
In the development of RNA interference (RNAi) therapeutics, merely selecting short, interfering RNA (siRNA) sequences that are complementary to the messenger RNA (mRNA) target does not guarantee target silencing. Current algorithms for selecting siRNAs rely on many parameters, one of which is asymmetry, often predicted through calculation of the relative thermodynamic stability of the two ends of the siRNA. However, we have previously shown that highly-active siRNA sequences are likely to have particular nucleotides at each 5’-end, independent of their thermodynamic asymmetry. Here, we describe an algorithm for predicting highly active siRNA sequences based only on these two asymmetry parameters. The algorithm uses end sequence nucleotide preferences and predicted thermodynamic stabilities, each weighted based on training data from the literature, to rank the probability that an siRNA sequence will have high or low activity. The algorithm successfully predicts weakly- and highly-active sequences for enhanced green fluorescent protein (EGFP) and protein kinase R (PKR). Use of these two parameters in combination improves the prediction of siRNA activity over current approaches for predicting asymmetry. Going forward, we anticipate that this approach to siRNA asymmetry prediction will be incorporated into the next generation of siRNA selection algorithms.
PMCID: PMC3924726  PMID: 24393396
asymmetry; siRNA; EGFP; PKR
10.  Time-scale separation: Michaelis and Menten's old idea, still bearing fruit 
The FEBS journal  2013;281(2):473-488.
Michaelis and Menten introduced to biochemistry the idea of time-scale separation, in which part of a system is assumed to be operating sufficiently fast compared to the rest that it may be assumed to have reached a steady state. This allows, in principle, the fast components to be eliminated, resulting in a simplified description of the system's behaviour. Similar ideas have been widely used in different areas of biology, including enzyme kinetics, protein allostery, receptor pharmacology, gene regulation and post-translational modification. However, the methods used have been independent and ad hoc. Here, we review the use of time-scale separation as a means to simplify the description of molecular complexity and discuss recent work which sets out a single framework which unifies these separate calculations. The framework offers new capabilities for mathematical analysis and helps to do justice to Michaelis and Menten's insights about individual enzymes in the context of multi-enzyme biological systems.
PMCID: PMC3991559  PMID: 24103070
11.  The Power of Integrating Kinetic Isotope Effects into the Formalism of the Michaelis-Menten Equation 
The FEBS journal  2013;281(2):489-497.
The final arbiter of enzyme mechanism is the ability to establish and test a kinetic mechanism. Isotope effects play a major role in expanding the scope and insight derived from the Michaelis-Menten equation. The integration of isotope effects into the formalism of the Michaelis-Menten equation began in the 1970s and has continued to this day. This review discusses a family of eukaryotic copper proteins that includes dopamine β-monooxygenase, tyramine β-monooxygenase, and peptidylglycine α-amidating enzyme, responsible for the synthesis of the neuro-active compounds, norepinephrine, octopamine and C-terminally carboxamidated peptides, respectively. Highlighted are results that show how combining kinetic isotope effects with initial rate parameters permits an evaluation of: (i) the order of substrate binding to multi-substrate enzymes; (ii) the magnitude of individual rate constants in complex, multi-step reactions; (iii) the identification of chemical intermediates; and (iv) the role of non-classical (tunneling) behavior in C–H activation.
PMCID: PMC4005594  PMID: 23937475
Steady-state kinetics; kinetic isotope effects; mechanism of enzyme action; mechanism of two copper monooxygenases; enzymatic C–H activation
12.  The Potential of Histone Deacetylase Inhibitors in Niemann–Pick Type C Disease 
The FEBS journal  2013;280(24):10.1111/febs.12505.
Niemann-Pick type C (NPC) disease is a fatal complex neurodegenerative lysosomal storage disorder caused by genetic mutations in either NPC1 (95% of patients) or NPC2 that decrease intracellular cholesterol trafficking resulting in accumulation of unesterified cholesterol and sphingolipids in lysosomal storage organelles. Unfortunately, treatment options for NPC disease are still very limited although miglustat, which inhibits glucosylceramide synthase, limiting ganglioside accumulation, has been approved for treatment of NPC disease. Here we discuss advances in understanding of NPC1 and its functions and several new strategies for interfering with cholesterol and sphingolipid accumulation in NPC1 null mice. We also describe several recent intriguing studies demonstrating that histone deacetylase inhibitors can correct cholesterol storage defects in human NPC1 mutant fibroblasts by increasing expression of the low transport activity NPC1 mutant protein. These studies might lead to development of new therapeutic approaches for treatment of NPC disease.
PMCID: PMC3857996  PMID: 23992240
Niemann–Pick type C disease; NPC1; NPC2; Cholesterol; Glycosphingolipids; HDAC
13.  GPR56 promotes myoblast fusion through SRE- and NFAT-mediated signaling but is not essential for muscle development in vivo 
The FEBS journal  2013;280(23):10.1111/febs.12529.
Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the G-protein coupled receptor 56 (GPR56) gene was transiently upregulated during the early fusion of human myoblasts. Human mutations in GPR56 cause the disease bilateral frontoparietal polymicrogyria (BFPP), however the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR56 in skeletal muscle. GPR56−/− myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, loss of GPR56 expression in muscle cells results in decreases or delays in the expression of MyoD, myogenin, and NFATc2. Our data suggest that these abnormalities result from decreased GPR56-mediated SRE and NFAT signaling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase (CK) compared to wildtype. In agreement with these findings, clinical data from 13 BFPP patients revealed mild serum CK increase in only 2 patients. In summary, targeted disruption of GPR56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR56 knockout mice and human patients suggests that other factors may compensate for the lack of this GPCR during muscle development and that the motor delay observed in these patients is likely not due to primary muscle abnormalities.
PMCID: PMC3877849  PMID: 24102982
GPR56; myoblast; skeletal muscle; dystroglycanopathies; SRE
14.  Thiol-Blocking Electrophiles Interfere with Labeling and Detection of Protein Sulfenic Acids 
The FEBS journal  2013;280(23):6150-6161.
Cellular exposure to reactive oxygen species induces the rapid oxidation of DNA, proteins, lipids, and other biomolecules. At the proteome level, cysteine thiol oxidation is a prominent post-translational process implicated in normal physiology and numerous pathologies. Methods for investigating protein oxidation include direct labeling with selective chemical probes and indirect tag-switch techniques. Common to both approaches is a chemical blocking of free thiols with reactive electrophiles to prevent post-lysis oxidation or other thiol-mediated cross-reactions. These reagents are used in large excess and their reactivity with cysteine sulfenic acid, a critical oxoform in numerous proteins, has not been investigated. Here we report the reactivity of three thiol-blocking electrophiles, iodoacetamide, N-ethylmaleimide, and methyl methanethiosulfonate, with protein sulfenic acid and dimedone, the structural core of many sulfenic acid probes. We demonstrate that covalent cysteine -SOR (product) species are susceptible to reduction by DTT, TCEP, and ascorbate, regenerating protein thiols, or in the case of ascorbate, more highly oxidized species. The implications of this reactivity on detection methods for protein sulfenic acids and S-nitrosothiols are discussed.
PMCID: PMC3928805  PMID: 24103186
Protein sulfenic acid; thiol blocking; S-nitrosation; dimedone; mass spectrometry
15.  Evasin-4, a tick-derived chemokine-binding protein with broad selectivity can be modified for use in preclinical disease models 
The Febs Journal  2013;280(19):4876-4887.
Rhipicephalus sanguineus, the common brown dog tick, produces several chemokine-binding proteins which are secreted into the host in its saliva to modulate the host response during feeding. Two of these demonstrate very restricted selectivity profiles. Here, we describe the characterization of the third, which we named Evasin-4. Evasin-4 was difficult to produce recombinantly using its native signal peptide in HEK cells, but expressed very well using the urokinase-type plasminogen activator signal peptide. Using SPR, Evasin-4 was shown to bind most CC chemokines. Investigation of the neutralization properties by inhibition of chemokine-induced chemotaxis showed that binding and neutralization did not correlate in all cases. Two major anomalies were observed: no binding was observed to CCL2 and CCL13, yet Evasin-4 was able to inhibit chemotaxis induced by these chemokines. Conversely, binding to CCL25 was observed, but Evasin-4 did not inhibit CCL25-induced chemotaxis. Size-exclusion chromatography confirmed that Evasin-4 forms a complex with CCL2 and CCL18. In accordance with the standard properties of unmodified small proteins, Evasin-4 was rapidly cleared following in vivo administration. To enhance the in vivo half-life and optimize its potential as a therapeutic agent, Fc fusions of Evasin-4 were created. Both the N- and C-terminal fusions were shown to retain binding activity, with the C-terminal fusion showing a modest reduction in potency.
PMCID: PMC4240464  PMID: 23910450
anti-inflammatory; binding protein; Fc fusion; hemokine; tick
16.  Analysis of the yeast short-term Crabtree effect and its origin 
The Febs Journal  2014;281(21):4805-4814.
The short-term Crabtree effect is defined as the immediate occurrence of aerobic alcoholic fermentation in response to provision of a pulse of excess sugar to sugar-limited yeast cultures. Here we have characterized ten yeast species with a clearly defined phylogenetic relationship. Yeast species were cultivated under glucose-limited conditions, and we studied their general carbon metabolism in response to a glucose pulse. We generated an extensive collection of data on glucose and oxygen consumption, and ethanol and carbon dioxide generation. We conclude that the Pichia,Debaryomyces,Eremothecium and Kluyveromyces marxianus yeasts do not exhibit any significant ethanol formation, while Kluyveromyces lactis behaves as an intermediate yeast, and Lachancea,Torulaspora,Vanderwaltozyma and Saccharomyces yeasts exhibit rapid ethanol accumulation. Based on the present data and our previous data relating to the presence of the long-term Crabtree effect in over 40 yeast species, we speculate that the origin of the short-term effect may coincide with the origin of the long-term Crabtree effect in the Saccharomycetales lineage, occurring ∼ 150 million years ago.
PMCID: PMC4240471  PMID: 25161062
aerobic fermentation; Crabtree effect; evolution; Saccharomyces; yeast
17.  Specific inhibition of PI3K p110δ inhibits CSF-1-induced macrophage spreading and invasive capacity 
The FEBS journal  2013;280(21):5228-5236.
Colony stimulating factor-1 (CSF-1) stimulates mononuclear phagocytic cell survival, growth and differentiation into macrophages through activation and autophosphorylation of the CSF-1 receptor (CSF-1R). We have previously demonstrated that CSF-1-induced phosphorylation of Y721 in the receptor kinase insert triggers its association with the p85 regulatory subunit of phosphoinositide 3′-kinase (PI3K). Binding of p85 PI3K to the CSF-1R pY721 motif activates the associated p110 PI3K catalytic subunit and stimulates spreading and motility in macrophages and their enhancement of tumor cell invasion. Here we show that pY721-based signaling is necessary for CSF-1-stimulated PtdIns(3,4,5)P (PIP3) production. While primary bone marrow-derived macrophages (BMM) and the immortalized bone marrow-derived macrophage cell line, M−/−.WT, express all three Class IA PI3K isoforms, p110δ predominates in the cell line. Treatment with p110δ specific inhibitors demonstrates that the hematopoietically enriched isoform, p110δ, mediates CSF-1 regulated spreading and invasion in macrophages. Thus GS-1101, a potent and selective p110δ inhibitor, may have therapeutic potential by targeting the infiltrative capacity of tumor-associated macrophages that is critical for their enhancement of tumor invasion and metastasis.
PMCID: PMC3773002  PMID: 23648053
macrophage; CSF-1R; PI3K; p110δ; GS-1101; invasion; DMSO
18.  What do we know about the Transient Receptor Potential Vanilloid 2 (TRPV2) ion channel? 
The FEBS journal  2013;280(21):5471-5487.
Transient receptor potential (TRP) ion channels are emerging as a new set of membrane proteins involved in a vast array of cellular processes and regulated by a large number of physical and chemical stimuli, which involves them with sensory cell physiology. The vanilloid TRP subfamily (TRPV) named after the vanilloid receptor 1 (TRPV1) consists of six members, and at least four of them (TRPV1-TRPV4) have been related to thermal sensation. One of the least characterized members of the TRP subfamily is TRPV2. Although initially characterized as a noxious heat sensor, TRPV2 now seems to have little to do with temperature sensing, but a much more complex physiological profile. Here we review the available information and research progress on the structure, physiology and pharmacology of TRPV2 in an attempt to shed some light on the physiological and pharmacological deorphanization of TRPV2.
PMCID: PMC3783526  PMID: 23615321
TRPV2; calcium signalling; somatosensation; neuroscience; immunology; cancer
19.  Role of Conformational Entropy in the Activity and Regulation of the catalytic subunit of Protein Kinase A 
The FEBS journal  2013;280(22):10.1111/febs.12462.
Protein kinase A (PKA) is the archetypical phosphokinase, sharing a catalytic core with the entire protein kinase superfamily. In eukaryotes, the ubiquitous location of PKA makes it one of the most important cellular signaling molecules, involved in a myriad of events. The catalytic-subunit of PKA (PKA-C) is one of the most studied enzymes and was the first kinase to be crystallized; however, the effects of ligand binding, post-translational modifications, and mutations on the activity of the kinase have been difficult to understand with only structural data. Here, we review our latest NMR studies on PKA-C, the results of which underscore the role of fast and slow conformational dynamics in the activation and inhibition of the kinase.
PMCID: PMC3821181  PMID: 23902454
Protein Kinase A; Allostery; Conformational Selection; Structural Dynamics; Conformational Entropy; NMR relaxation
20.  The Transporter-Opsin-G protein-coupled receptor (TOG) Superfamily 
The FEBS journal  2013;280(22):10.1111/febs.12499.
Visual Rhodopsins (VR) are recognized members of the large and diverse family of G protein-coupled receptors (GPCRs), but their evolutionary origin and relationships to other proteins, are not known. In an earlier publication (Shlykov et al., 2012), we characterized the 4-Toulene Sulfonate Uptake Permease (TSUP) family of transmembrane proteins, showing that these 7 or 8 TMS proteins arose by intragenic duplication of a 4 TMS-encoding gene, sometimes followed by loss of a terminal TMS. In this study, we show that the TSUP, GPCR and Microbial Rhodopsin (MR) families are related to each other and to six other currently recognized transport protein families. We designate this superfamily the Transporter-Opsin-G protein-coupled receptor (TOG) Superfamily. Despite their 8 TMS origins, members of most constituent families exhibit 7 TMS topologies that are well conserved, and these arose by loss of either the N-terminal (more frequent) or the C-terminal (less frequent) TMS, depending on the family. Phylogenetic analyses revealed familial relationships within the superfamily and protein relationships within each of the nine families. The statistical analyses leading to the conclusion of homology were confirmed using HMMs, Pfam, and 3D superimpositions. Proteins functioning by dissimilar mechanisms (channels, primary active transporters, secondary active transporters, group translocators and receptors) are interspersed on a phylogenetic tree of the TOG superfamily, suggesting that changes in the transport and energy-coupling mechanisms occurred multiple times during the evolution of this superfamily.
PMCID: PMC3832197  PMID: 23981446
Transport proteins; channels; secondary carriers; receptors; rhodopsin
21.  Protein crystallography for aspiring crystallographers or how to avoid pitfalls and traps in macromolecular structure determination 
The FEBS journal  2013;280(22):5705-5736.
The number of macromolecular structures deposited in the Protein Data Bank now approaches 100 000, with the vast majority of them determined by crystallographic methods. Thousands of papers describing such structures have been published in the scientific literature, and 20 Nobel Prizes in chemistry or medicine have been awarded for discoveries based on macromolecular crystallography. New hardware and software tools have made crystallography appear to be an almost routine (but still far from being analytical) technique and many structures are now being determined by scientists with very limited experience in the practical aspects of the field. However, this apparent ease is sometimes illusory and proper procedures need to be followed to maintain high standards of structure quality. In addition, many noncrystallographers may have problems with the critical evaluation and interpretation of structural results published in the scientific literature. The present review provides an outline of the technical aspects of crystallography for less experienced practitioners, as well as information that might be useful for users of macromolecular structures, aiming to show them how to interpret (but not overinterpret) the information present in the coordinate files and in their description. A discussion of the extent of information that can be gleaned from the atomic coordinates of structures solved at different resolution is provided, as well as problems and pitfalls encountered in structure determination and interpretation.
PMCID: PMC4080831  PMID: 24034303
data collection and processing; electron density maps; protein crystallography; structure refinement; structure solution; structure quality; structure validation
22.  Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene-modified DNA by fluorescence methods 
The FEBS journal  2012;279(20):3965-3980.
The impact of bulky carcinogen-DNA adducts positioned at or near recognition sites (CpG) of eukaryotic DNA methyltransferases on their catalytic activities is poorly understood. In this work we have employed site-specifically modified 30-mer oligodeoxyribonucleotides containing stereoisomeric benzo[a]pyrene diol epoxide (B[a]PDE)-derived guanine (B[a]PDE-N2-dG) or adenine (B[a]PDE-N6-dA) adducts of different conformations as substrates of catalytic domain of murine Dnmt3a (Dnmt3a-CD). The fluorescence of these lesions was used to examine interactions between Dnmt3a-CD and DNA. In B[a]PDE-DNA•Dnmt3a-CD complexes the intensity of fluorescence of the covalently bound B[a]PDE residues is enhanced relative to the protein-free value when the B[a]PDE is positioned in the minor groove ((+)- and (−)-trans-B[a]PDE-N2-dG adducts in the CpG site) and when it is intercalated on the 5′-side of the CpG site ((+)-trans- B[a]PDE-N6-dA adduct). The fluorescence of B[a]PDE-modified DNA•Dnmt3a-CD complexes exhibits only small changes when the B[a]PDE is intercalated with base displacement in (+)- and (−)-cis- B[a]PDE-N2-dG adducts and without base displacement in the (−)-trans- B[a]PDE-N6-dA adduct. The initial rates of methylation were significantly reduced by the minor groove trans-B[a]PDE-N2-dG adducts regardless of their position in the substrate and by the intercalated cis-B[a]PDE-N2-dG adducts within the CpG site. The observed changes in fluorescence and methylation rates are consistent with the flipping of the target cytosine and a catalytic loop motion within the DNA•Dnmt3a-CD complexes. In the presence of the regulatory factor Dnmt3L, an enhancement of the methylation rates and fluorescence were observed which is consistent with a Dnmt3L-mediated displacement of the catalytic loop towards the CpG site.
PMCID: PMC4208501  PMID: 22913541
Benzo[a]pyrene-DNA; minor groove; intercalation; fluorescence; methylation
23.  Crystal Structures of Apparent Saccharide Sensors from Histidine Kinase Receptors Prevalent in a Human Gut Symbiont 
The FEBS journal  2014;281(18):4263-4279.
The adult human gut presents a complicated ecosystem where host-bacterium symbiosis plays an important role. Bacteroides thetaiotaomicron is a predominant member of the gut microflora, providing the human digestive tract with a large number of glycolytic enzymes. Expression of many of these enzymes appears to be controlled by histidine kinase receptors that are fused into unusual hybrid two-component systems that share homologous periplasmic sensor domains. These sensor domains belong to the third most populated (HK3) family based on a previous bioinformatics analysis of predicted histidine kinase sensors. Here, we present crystal structures of two sensor domains representative of the HK3 family. Each sensor is folded into three domains: two seven-bladed β-propeller domains and one β-sandwich domain. Both sensors form dimers in crystals and one sensor appears to be physiologically relevant. The folding characteristics in the individual domains, the domain organization, and the oligomeric architecture are all unique to the HK3 sensors. The sequence analysis of the HK3 sensors indicates that these sensors are shared among other signaling molecules, implying a combinatorial molecular evolution.
PMCID: PMC4169315  PMID: 24995510
beta-propeller domain; beta-sandwich domain; hybrid two-component system; saccharide sensing; signal transduction; dimerization
24.  Mechanisms for ATP-dependent chromatin remodelling: The means to the end 
The FEBS journal  2011;278(19):3579-3595.
Chromatin remodelling is the ATP-dependent change in nucleosome organisation driven by Snf2 family ATPases. The biochemistry of this process depends on the behaviours of ATP dependent motor proteins and their dynamic nucleosome substrates, which brings significant technical and conceptual challenges. Steady progress has been made in characterizing the polypeptides that these enzymes are comprised of. Divergence in the sequences of different subfamilies of Snf2 related proteins suggests that the motors are adapted for different functions. Recently structural insights have suggested that the Snf2 ATPase acts as a context-sensitive DNA translocase. This may have arisen as a means to enable efficient access to DNA in the high density of the eukaryotic nucleus. How the enzymes engage nucleosomes and how the network of non-covalent interactions within the nucleosome respond to the force applied remains unclear, and it remains prudent to recognise the potential for both DNA distortions and dynamics within the underlying histone octamer structure.
PMCID: PMC4162296  PMID: 21810178
25.  Evidence for the Slow Reaction of Hypoxia-Inducible Factor Prolyl Hydroxylase 2 with Oxygen 
The FEBS journal  2010;277(19):4089-4099.
The response of animals to hypoxia is mediated by the hypoxia-inducible transcription factor (HIF). Human HIF is regulated by four Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases: Prolyl hydroxylase domain enzymes (PHDs or EGLNs) 1–3 catalyse hydroxylation of two prolyl-residues in HIF, triggering its degradation by the proteasome. Factor inhibiting HIF (FIH) catalyses hydroxylation of an asparagine-residue in HIF, inhibiting its transcriptional activity. Collectively, the HIF hydroxylases negatively regulate HIF in response to increasing oxygen concentration. Prolyl hydroxylase domain 2 (PHD2) is the most important oxygen sensor in human cells; however the underlying kinetic basis of the oxygen sensing function of PHD2 is unclear. We report analyses of the reaction of PHD2 with oxygen. Chemical quench/mass spectrometry experiments showed that reaction of a complex of PHD2, Fe(II), 2OG and the C-terminal oxygen-dependent degradation domain of HIF-α (CODD) with oxygen to form hydroxylated CODD and succinate is much slower (~100 fold) than for other similarly studied 2OG oxygenases. Stopped flow/UV-visible spectroscopy experiments showed that the reaction produces a relatively stable species absorbing at 320nm; Mössbauer spectroscopic experiments implied that this species is likely not a Fe(IV)=O intermediate, as observed for other 2OG oxygenases. Overall the results suggest that, at least compared to other studied 2OG oxygenases, PHD2 reacts relatively slowly with oxygen, a property that may be associated with its function as an oxygen sensor.
PMCID: PMC4160827  PMID: 20840591
Hypoxia Inducible Factor; 2-Oxoglutarate; Oxygen; Oxygenase; Prolyl Hydroxylase; Spectroscopy

Results 1-25 (406)