Publications by authors named "Denis Titov"

18 Publications

  • Page 1 of 1

A Cellular Mechanism to Detect and Alleviate Reductive Stress.

Cell 2020 Oct 16;183(1):46-61.e21. Epub 2020 Sep 16.

Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley CA 94720, USA; Howard Hughes Medical Institute, University of California at Berkeley, Berkeley CA 94720, USA; California Institute for Quantitative Biosciences (QB3), University of California at Berkeley, Berkeley, CA 94720, USA. Electronic address:

Metazoan organisms rely on conserved stress response pathways to alleviate adverse conditions and preserve cellular integrity. Stress responses are particularly important in stem cells that provide lifetime support for tissue formation and repair, but how these protective systems are integrated into developmental programs is poorly understood. Here we used myoblast differentiation to identify the E3 ligase CUL2 and its substrate FNIP1 as core components of the reductive stress response. Reductive stress, as caused by prolonged antioxidant signaling or mitochondrial inactivity, reverts the oxidation of invariant Cys residues in FNIP1 and allows CUL2 to recognize its target. The ensuing proteasomal degradation of FNIP1 restores mitochondrial activity to preserve redox homeostasis and stem cell integrity. The reductive stress response is therefore built around a ubiquitin-dependent rheostat that tunes mitochondrial activity to redox needs and implicates metabolic control in coordination of stress and developmental signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.08.034DOI Listing
October 2020

Distinct mitochondrial defects trigger the integrated stress response depending on the metabolic state of the cell.

Elife 2020 05 28;9. Epub 2020 May 28.

Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, United States.

Mitochondrial dysfunction is associated with activation of the integrated stress response (ISR) but the underlying triggers remain unclear. We systematically combined acute mitochondrial inhibitors with genetic tools for compartment-specific NADH oxidation to trace mechanisms linking different forms of mitochondrial dysfunction to the ISR in proliferating mouse myoblasts and in differentiated myotubes. In myoblasts, we find that impaired NADH oxidation upon electron transport chain (ETC) inhibition depletes asparagine, activating the ISR via the eIF2α kinase GCN2. In myotubes, however, impaired NADH oxidation following ETC inhibition neither depletes asparagine nor activates the ISR, reflecting an altered metabolic state. ATP synthase inhibition in myotubes triggers the ISR via a distinct mechanism related to mitochondrial inner-membrane hyperpolarization. Our work dispels the notion of a universal path linking mitochondrial dysfunction to the ISR, instead revealing multiple paths that depend both on the nature of the mitochondrial defect and on the metabolic state of the cell.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.49178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7255802PMC
May 2020

Transgenerational Sterility of Piwi Mutants Represents a Dynamic Form of Adult Reproductive Diapause.

Cell Rep 2018 04;23(1):156-171

Department of Genetics, University of North Carolina, Chapel Hill, NC 27599, USA; Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA; Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599, USA. Electronic address:

Environmental stress can induce adult reproductive diapause, a state of developmental arrest that temporarily suspends reproduction. Deficiency for C. elegans Piwi protein PRG-1 results in strains that reproduce for many generations but then become sterile. We found that sterile-generation prg-1/Piwi mutants typically displayed pronounced germ cell atrophy as L4 larvae matured into 1-day-old adults. Atrophied germlines spontaneously reproliferated across the first days of adulthood, and this was accompanied by fertility for day 2-4 adults. Sterile day 5 prg-1 mutant adults remained sterile indefinitely, but providing an alternative food source could restore their fertility. Our data imply that late-generation prg-1 mutants experience a dynamic form of adult reproductive diapause, promoted by stress response, cell death, and RNAi pathways, where delayed fertility and reproductive quiescence represent parallel adaptive developmental outcomes. This may occur in response to a form of "heritable stress" that is transmitted by gametes and epigenetic in nature.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.03.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5918633PMC
April 2018

The Mechanism of Low-Temperature Oxidation of Carbon Monoxide by Oxygen over the PdCl₂-CuCl₂/γ-Al₂O₃ Nanocatalyst.

Nanomaterials (Basel) 2018 Apr 3;8(4). Epub 2018 Apr 3.

Moscow Technological University, Institute of Fine Chemical Technology, Department of General Chemical Technology, Moscow 119571, Russia.

The state of palladium and copper on the surface of the PdCl₂-CuCl₂/γ-Al₂O₃ nanocatalyst for the low-temperature oxidation of CO by molecular oxygen was studied by various spectroscopic techniques. Using X-ray absorption spectroscopy (XAS), powder X-ray diffraction (XRD), and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), freshly prepared samples of the catalyst were studied. The same samples were also evaluated after interaction with CO, O₂, and H₂O vapor in various combinations. It was shown that copper exists in the form of Cu₂Cl(OH)₃ (paratacamite) nanophase on the surface of the catalyst. No palladium-containing crystalline phases were identified. Palladium coordination initially is comprised of four chlorine atoms. It was shown by XAS that this catalyst is not capable of oxidizing CO at room temperature in the absence of H₂O and O₂ over 12 h. Copper(II) and palladium(II) are reduced to Cu(I) and Pd(I,0) species, respectively, in the presence of CO and H₂O vapor (without O₂). It was found by DRIFTS that both linear (2114 cm, 1990 cm) and bridging (1928 cm) forms of coordinated CO were formed upon adsorption onto the catalyst surface. Moreover, the formation of CO₂ was detected upon the interaction of the coordinated CO with oxygen. The kinetics of CO oxidation was studied at 18-38 °C at an atmospheric pressure for CO, O₂, N₂, and H₂O (gas) mixtures in a flow reactor (steady state conditions).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/nano8040217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923547PMC
April 2018

Anti-inflammaging effects of human alpha-1 antitrypsin.

Aging Cell 2018 02 17;17(1). Epub 2017 Oct 17.

Department of Molecular Genetics & Microbiology, University of Florida, Gainesville, FL, USA.

Inflammaging plays an important role in most age-related diseases. However, the mechanism of inflammaging is largely unknown, and therapeutic control of inflammaging is challenging. Human alpha-1 antitrypsin (hAAT) has immune-regulatory, anti-inflammatory, and cytoprotective properties as demonstrated in several disease models including type 1 diabetes, arthritis, lupus, osteoporosis, and stroke. To test the potential anti-inflammaging effect of hAAT, we generated transgenic Drosophila lines expressing hAAT. Surprisingly, the lifespan of hAAT-expressing lines was significantly longer than that of genetically matched controls. To understand the mechanism underlying the anti-aging effect of hAAT, we monitored the expression of aging-associated genes and found that aging-induced expressions of Relish (NF-ĸB orthologue) and Diptericin were significantly lower in hAAT lines than in control lines. RNA-seq analysis revealed that innate immunity genes regulated by NF-kB were significantly and specifically inhibited in hAAT transgenic Drosophila lines. To confirm this anti-inflammaging effect in human cells, we treated X-ray-induced senescence cells with hAAT and showed that hAAT treatment significantly decreased the expression and maturation of IL-6 and IL-8, two major factors of senescence-associated secretory phenotype. Consistent with results from Drosophila,RNA-seq analysis also showed that hAAT treatment significantly inhibited inflammation related genes and pathways. Together, our results demonstrated that hAAT significantly inhibited inflammaging in both Drosophila and human cell models. As hAAT is a FDA-approved drug with a confirmed safety profile, this novel therapeutic potential may make hAAT a promising candidate to combat aging and aging-related diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/acel.12694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5770780PMC
February 2018

A genetically encoded tool for manipulation of NADP/NADPH in living cells.

Nat Chem Biol 2017 Oct 7;13(10):1088-1095. Epub 2017 Aug 7.

Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.

The redox coenzymes NADH and NADPH are broadly required for energy metabolism, biosynthesis and detoxification. Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes, a holistic understanding of the regulation and compartmentalization of NADH- and NADPH-dependent pathways is lacking, partly because of a lack of tools with which to investigate these processes in living cells. We have previously reported the use of the naturally occurring Lactobacillus brevis HO-forming NADH oxidase (LbNOX) as a genetic tool for manipulation of the NAD/NADH ratio in human cells. Here, we present triphosphopyridine nucleotide oxidase (TPNOX), a rationally designed and engineered mutant of LbNOX that is strictly specific to NADPH. We characterized the effects of TPNOX expression on cellular metabolism and used it in combination with LbNOX to show how the redox states of mitochondrial NADPH and NADH pools are connected.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio.2454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605434PMC
October 2017

Complementation of mitochondrial electron transport chain by manipulation of the NAD+/NADH ratio.

Science 2016 Apr 7;352(6282):231-5. Epub 2016 Apr 7.

Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA. Department of Systems Biology, Harvard Medical School, Boston, MA, USA. Broad Institute, Cambridge, MA, USA.

A decline in electron transport chain (ETC) activity is associated with many human diseases. Although diminished mitochondrial adenosine triphosphate production is recognized as a source of pathology, the contribution of the associated reduction in the ratio of the amount of oxidized nicotinamide adenine dinucleotide (NAD(+)) to that of its reduced form (NADH) is less clear. We used a water-forming NADH oxidase from Lactobacillus brevis (LbNOX) as a genetic tool for inducing a compartment-specific increase of the NAD(+)/NADH ratio in human cells. We used LbNOX to demonstrate the dependence of key metabolic fluxes, gluconeogenesis, and signaling on the cytosolic or mitochondrial NAD(+)/NADH ratios. Expression of LbNOX in the cytosol or mitochondria ameliorated proliferative and metabolic defects caused by an impaired ETC. The results underscore the role of reductive stress in mitochondrial pathogenesis and demonstrate the utility of targeted LbNOX for direct, compartment-specific manipulation of redox state.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aad4017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850741PMC
April 2016

Homozygous deletion in MICU1 presenting with fatigue and lethargy in childhood.

Neurol Genet 2016 Apr 3;2(2):e59. Epub 2016 Mar 3.

Wellcome Trust Centre for Mitochondrial Research (D.L.-S., H.G., J.D., A.P., R.W.T., P.Y.-W.-M., R.H., P.F.C.), Institute of Genetic Medicine (D.L.-S., H.G., J.D., A.P., P.Y.-W.-M., R.H.), and Institute of Neuroscience (R.W.T.), Newcastle University, Newcastle upon Tyne, United Kingdom; Howard Hughes Medical Institute (K.J.K., D.T., V.K.M.), Department of Molecular Biology, Massachusetts General Hospital, Boston, MA; Department of Paediatric Neurology (A.-M.C., K.P.), The General Infirmary, Leeds, United Kingdom; Department of Child Neurology (V.R.), The Royal Victoria Infirmary, Newcastle upon Tyne Hospitals NHS Trust, Newcastle upon Tyne, United Kingdom; Department of Systems Biology (V.K.M.), Harvard Medical School, Boston, MA; Broad Institute (V.K.M.), Cambridge, MA; Department of Clinical Neurosciences (P.F.C.), University of Cambridge; and MRC Mitochondrial Biology Unit (P.F.C.), Cambridge Biomedical Campus, United Kingdom.

Objective: To define the mechanism responsible for fatigue, lethargy, and weakness in 2 cousins who had a normal muscle biopsy.

Methods: Exome sequencing, long-range PCR, and Sanger sequencing to identify the pathogenic mutation. Functional analysis in the patient fibroblasts included oxygen consumption measurements, extracellular acidification studies, Western blotting, and calcium imaging, followed by overexpression of the wild-type protein.

Results: Analysis of the exome sequencing depth revealed a homozygous deletion of exon 1 of MICU1 within a 2,755-base pair deletion. No MICU1 protein was detected in patient fibroblasts, which had impaired mitochondrial calcium uptake that was rescued through the overexpression of the wild-type allele.

Conclusions: MICU1 mutations cause fatigue and lethargy in patients with normal mitochondrial enzyme activities in muscle. The fluctuating clinical course is likely mediated through the mitochondrial calcium uniporter, which is regulated by MICU1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1212/NXG.0000000000000059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4830195PMC
April 2016

Trimodal Control of Ion-Transport Activity on Cyclo-oligo-(1→6)-β-D-glucosamine-Based Artificial Ion-Transport Systems.

Chemistry 2015 Nov 8;21(48):17445-52. Epub 2015 Oct 8.

Department of Chemistry, Indian Institute of Science Education and Research Pune, Dr. Homi Bhabha Road, Pashan, Pune 411008, Maharashtra (India), Fax: (+91) 20-25899790.

Cyclo-oligo-(1→6)-β-D-glucosamines functionalized with hydrophobic tails are reported as a new class of transmembrane ion-transport system. These macrocycles with hydrophilic cavities were introduced as an alternative to cyclodextrins, which are supramolecular systems with hydrophobic cavities. The transport activities of these glycoconjugates were manipulated by altering the oligomericity of the macrocycles, as well as the length and number of attached tails. Hydrophobic tails of 3 different sizes were synthesized and coupled with each glucosamine scaffold through the amide linkage to obtain 18 derivatives. The ion-transport activity increased from di- to tetrameric glucosamine macrocycles, but decreased further when flexible pentameric glucosamine was introduced. The ion-transport activity also increased with increasing length of attached linkers. For a fixed length of linkers, the transport activity decreased when the number of such tails was reduced. All glycoconjugates displayed a uniform anion-selectivity sequence: Cl(-) >Br(-) >I(-) . From theoretical studies, hydrogen bonding between the macrocycle backbone and the anion bridged through water molecules was observed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chem.201502656DOI Listing
November 2015

Covalent modification of a cysteine residue in the XPB subunit of the general transcription factor TFIIH through single epoxide cleavage of the transcription inhibitor triptolide.

Angew Chem Int Ed Engl 2015 Feb 12;54(6):1859-63. Epub 2014 Dec 12.

Department of Pharmacology, Johns Hopkins School of Medicine, 725 North Wolfe Street, Hunterian Building, Room 516, Baltimore, MD 21205 (USA).

Triptolide is a key component of the traditional Chinese medicinal plant Thunder God Vine and has potent anticancer and immunosuppressive activities. It is an irreversible inhibitor of eukaryotic transcription through covalent modification of XPB, a subunit of the general transcription factor TFIIH. Cys342 of XPB was identified as the residue that undergoes covalent modification by the 12,13-epoxide group of triptolide. Mutation of Cys342 of XPB to threonine conferred resistance to triptolide on the mutant protein. Replacement of the endogenous wild-type XPB with the Cys342Thr mutant in a HEK293T cell line rendered it completely resistant to triptolide, thus validating XPB as the physiologically relevant target of triptolide. Together, these results deepen our understanding of the interaction between triptolide and XPB and have implications for the future development of new analogues of triptolide as leads for anticancer and immunosuppressive drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201408817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4314353PMC
February 2015

Cyclo-oligo-(1 → 6)-β-D-glucosamine based artificial channels for tunable transmembrane ion transport.

Chem Commun (Camb) 2014 May;50(41):5514-6

Department of Chemistry, Indian Institute of Science Education and Research Pune, Maharashtra 411008, India.

Unimolecular ion channels were designed by functionalization of a new type of cyclic oligosaccharides, cyclo-oligo-(1 → 6)-β-d-glucosamines, with pentabutylene glycol chains. Their ion transporting activity was tuned by varying oligomericity. A halide selectivity sequence, Cl(-) > Br(-) > I(-) was observed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c3cc49490jDOI Listing
May 2014

Synthesis of multivalent carbohydrate-centered glycoclusters as nanomolar ligands of the bacterial lectin LecA from Pseudomonas aeruginosa.

Chemistry 2013 Jul 11;19(28):9272-85. Epub 2013 Jun 11.

Laboratory of Glycoconjugate Chemistry, N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect 47, 119991 Moscow, Russia.

A family of fifteen glycoclusters based on a cyclic oligo-(1→6)-β-D-glucosamine core has been designed as potential inhibitors of the bacterial lectin LecA with various valencies (from 2 to 4) and linkers. Evaluation of their binding properties towards LecA has been performed by a combination of hemagglutination inhibition assays (HIA), enzyme-linked lectin assays (ELLA), and isothermal titration microcalorimetry (ITC). Divalent ligands displayed dissociation constants in the sub-micromolar range and tetravalent ligands displayed low nanomolar affinities for this lectin. The influence of the linker could also be demonstrated; aromatic moieties are the best scaffolds for binding to the lectin. The affinities observed in vitro were then correlated with molecular models to rationalize the possible binding modes of these glycoclusters with the bacterial lectin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chem.201300135DOI Listing
July 2013

Stereochemistry of intramolecular cyclization of tetra-β-(1→6)-D-glucosamines and related tetrasaccharides: the role of the conformational stereocontrol and the neighboring group participation.

Carbohydr Res 2013 Nov 20;381:161-78. Epub 2012 Dec 20.

Laboratory of Glycoconjugate Chemistry, N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect 47, 119991 Moscow, Russia.

The effect of reaction conditions, the nature of a leaving group, and a substituent at C-2 in the glycosylating monosaccharide on the stereochemical outcome of cyclization of linear tetra-β-(1→6)-d-glucosamines and some 'mixed' tetrasaccharides comprising glucose and glucosamine residues has been examined. Toluene and nitrile solvents improved the β-stereoselectivity of cyclization, however, the overall efficiency of the formation of cyclic products in these solvents was lower than that in dichloromethane. The use of bromide or pentenyl glycoside as leaving groups instead of the thioglycoside did not increase the β-stereoselectivity. Replacement of the N-phthaloyl group in the glycosylating unit by N-Troc one did not affect the stereoselectivity of cyclization, while the tetrasaccharides, which contained 2-O-benzoyl glucose instead of glucosamine as the glycosyl donor moiety, were found to provide β-linked cyclic products exclusively. Using this finding, two cyclic tetrasaccharides with alternate or adjacent arrangement of two glucose and two glucosamine units have been efficiently synthesized. These cycles were intended for the preparation of divalent glycoclusters with different ligand orientation. The difference in the stereoselectivity of cyclization of glucose and glucosamine precursors was accounted for by more effective anchimeric participation of the O-benzoyl group as compared to N-Phth and N-Troc counterparts that was confirmed by calculations of the stabilization energy and rotational barriers around C2-O/N bond in the corresponding glycosyl cations. Only the 2-O-benzoylated glycosyl cation proved to be able to form a stabilized dioxalenium intermediate, which provides stereospecific β-glycosylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.carres.2012.12.005DOI Listing
November 2013

Identification and validation of protein targets of bioactive small molecules.

Bioorg Med Chem 2012 Mar 20;20(6):1902-9. Epub 2011 Dec 20.

Department of Pharmacology, Johns Hopkins University School of Medicine, MD, USA.

Identification and validation of protein targets of bioactive small molecules is an important problem in chemical biology and drug discovery. Currently, no single method is satisfactory for this task. Here, we provide an overview of common methods for target identification and validation that historically were most successful. We have classified for the first time the existing methods into two distinct and complementary types, the 'top-down' and 'bottom-up' approaches. In a typical top-down approach, the cellular phenotype is used as a starting point and the molecular target is approached through systematic narrowing down of possibilities by taking advantage of the detailed existing knowledge of cellular pathways and processes. In contrast, the bottom-up approach entails the direct detection and identification of the molecular targets using affinity-based or genetic methods. A special emphasis is placed on target validation, including correlation analysis and genetic methods, as this area is often ignored despite its importance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2011.11.070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3714012PMC
March 2012

NMR and conformational studies of linear and cyclic oligo-(1→6)-β-D-glucosamines.

Carbohydr Res 2011 Nov 5;346(15):2499-510. Epub 2011 Sep 5.

Laboratory of Glycoconjugate Chemistry, N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect 47, 119991 Moscow, Russia.

The conformational behavior of a series of linear and cyclic oligo-(1→6)-β-D-glucosamines and their N-acetylated derivatives, which are related to fragments of natural poly-N-acetylglucosamine, was studied by theoretical molecular modeling and experimental determination of transglycosidic vicinal coupling constants (3)J(C,H) and (3)J(H,H). Molecular dynamics simulations were performed under several types of conditions varying in the consideration of ionization of amino groups, solvent effect, and temperature. Neural network clustering and asphericity calculations were performed on the basis of molecular dynamics data. It was shown that disaccharide fragments in the studied linear oligosaccharides were not rigid, and tended to have several conformers, thus determining the overall twisted shape with helical elements. In addition, it was found that the behavior of C5-C6 bond depended significantly upon the simulation conditions. The cyclic di-, tri-, and tetrasaccharides mostly had symmetrical ring-shaped conformations. The larger cycles tended to adopt more complicated shapes, and the conformational behavior of their disaccharide fragments was close to that in the linear oligosaccharides.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.carres.2011.08.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3201778PMC
November 2011

XPB, a subunit of TFIIH, is a target of the natural product triptolide.

Nat Chem Biol 2011 Mar 30;7(3):182-8. Epub 2011 Jan 30.

Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Triptolide (1) is a structurally unique diterpene triepoxide isolated from a traditional Chinese medicinal plant with anti-inflammatory, immunosuppressive, contraceptive and antitumor activities. Its molecular mechanism of action, however, has remained largely elusive to date. We report that triptolide covalently binds to human XPB (also known as ERCC3), a subunit of the transcription factor TFIIH, and inhibits its DNA-dependent ATPase activity, which leads to the inhibition of RNA polymerase II-mediated transcription and likely nucleotide excision repair. The identification of XPB as the target of triptolide accounts for the majority of the known biological activities of triptolide. These findings also suggest that triptolide can serve as a new molecular probe for studying transcription and, potentially, as a new type of anticancer agent through inhibition of the ATPase activity of XPB.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio.522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3622543PMC
March 2011

Regulator of Ras depalmitoylation and retrograde trafficking: a new hat for FKBP.

Mol Cell 2011 Jan;41(2):131-3

Department of Pharmacology, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

In this issue of Molecular Cell, Ahearn et al. (2011) identified FKBP12 as a novel regulator of Ras signaling through its modulation of depalmitoylation of H-Ras and its recycling from plasma membrane to the Golgi.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2011.01.009DOI Listing
January 2011

Analyzing protein kinase dynamics in living cells with FRET reporters.

Methods 2006 Nov;40(3):279-86

Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Genetically encoded reporters based on fluorescence resonance energy transfer (FRET) are being developed for analyzing spatiotemporal dynamics of kinase activities in living cells, as the activities of this class of enzymes are often dynamically regulated and spatially compartmentalized within specific signaling context. Here we describe a general modular design and engineering strategies for the development of activity reporters for kinases of interest, using A-kinase activity reporter (AKAR) as an illustrative example. Discussed here are basic structure of such reporters, design considerations, reporter gene construction, cellular and in vitro characterization. Strategies for improving specificity, dynamic range or sensitivity, reversibility and integrity of the reporter as well as basic methods for live-cell time-lapse imaging using these reporters are summarized. Discussion of using this approach in the study of MAPK cascades is also provided. These FRET-based kinase activity reporters, along with analogous probes based on alternative designs, provide real-time tracking of kinase dynamics with subcellular resolution, which should complement other methods and offer great opportunities to delineate the molecular mechanisms underlying the complex regulation of kinases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymeth.2006.06.013DOI Listing
November 2006