78 results on '"Elena Kudryashova"'
Search Results
2. Liposomal Forms of Fluoroquinolones and Antifibrotics Decorated with Mannosylated Chitosan for Inhalation Drug Delivery
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Irina Le-Deygen, Anastasia Safronova, Polina Mamaeva, Yana Khristidis, Ilya Kolmogorov, Anna Skuredina, Peter Timashev, and Elena Kudryashova
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liposomes ,antibacterial drugs ,inhaled drug delivery ,antifibrotic drugs ,spectroscopy ,chitosan ,Pharmacy and materia medica ,RS1-441 - Abstract
The severe course of COVID-19 leads to the long-terming pulmonary diseases, such as bacterial pneumonia and post-COVID-19 pulmonary fibrosis. Thus, the essential task of biomedicine is a design of new effective drug formulations, including those for inhalation administration. In this work, we propose an approach to the creation of lipid–polymer delivery systems for fluoroquinolones and pirfenidone based on liposomes of various compositions decorated with mucoadhesive mannosylated chitosan. A generalizing study on the physicochemical patterns of the interactions of drugs with bilayers of various compositions was carried out, and the main binding sites were identified. The role of the polymer shell in the stabilization of vesicles and the delayed release of the contents has been demonstrated. For the liquid–polymer formulation of moxifloxacin, a prolonged accumulation of the drug in lung tissues was found after a single endotracheal administration to mice, significantly exceeding the control intravenous and endotracheal administration of the drug.
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- 2023
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3. Chitosan-Covered Calcium Phosphate Particles Co-Loaded with Superoxide Dismutase 1 and ACE Inhibitor: Development, Characterization and Effect on Intraocular Pressure
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Ekaterina Popova, Olesya Matveeva, Olga Beznos, Victoria Tikhomirova, Elena Kudryashova, Yuri Grigoriev, Natalia Chesnokova, and Olga Kost
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calcium phosphate nanoparticles ,chitosan ,superoxide dismutase 1 ,angiotensin-converting enzyme inhibitor ,intraocular pressure ,eye diseases ,Pharmacy and materia medica ,RS1-441 - Abstract
Improvement of the efficiency of drug penetration into the eye tissues is still an actual problem in ophthalmology. One of the most promising solutions is drug encapsulation in carriers capable of overcoming the cornea/sclera tissue barrier. Formulations on the base of antioxidant enzyme, superoxide dismutase 1 (SOD1), and an inhibitor of angiotensin-converting enzyme, enalaprilat, were prepared by simultaneous inclusion of both drugs into calcium phosphate (CaP) particles in situ with subsequent covering of the particles with 5 kDa chitosan. The formulations obtained were characterized by dynamic light scattering and scanning electron microscopy. Hybrid CaP-chitosan particles co-loaded with SOD1 and enalaprilat had a mean hydrodynamic diameter of 120–160 nm and ζ-potential +20 ± 1 mV. The percentage of the inclusion of SOD1 and enalaprilat in hybrid particles was 30% and 56%, respectively. The ability of SOD1 and enalaprilat to reduce intraocular pressure (IOP) was examined in vivo in normotensive Chinchilla rabbits. It was shown that topical instillations of SOD1/enalaprilat co-loaded hybrid particles were much more effective in decreasing IOP compared to free enzyme or free enalaprilat and even to the same particles that contained a single drug. Thus, the proposed formulations demonstrate potential as prospective therapeutic agents for the treatment of glaucoma.
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- 2023
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4. Spectroscopy Study of Albumin Interaction with Negatively Charged Liposome Membranes: Mutual Structural Effects of the Protein and the Bilayers
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Daria Tretiakova, Maria Kobanenko, Irina Le-Deygen, Ivan Boldyrev, Elena Kudryashova, Natalia Onishchenko, and Elena Vodovozova
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protein adsorption ,albumin ,liposome–protein complexes ,fluorescence spectroscopy ,ATR-FTIR ,Chemical technology ,TP1-1185 ,Chemical engineering ,TP155-156 - Abstract
Liposomes as drug carriers are usually injected into the systemic circulation where they are instantly exposed to plasma proteins. Liposome–protein interactions can affect both the stability of liposomes and the conformation of the associated protein leading to the altered biodistribution of the carrier. In this work, mutual effects of albumin and liposomal membrane in the course of the protein’s adsorption were examined in terms of quantity of bound protein, its structure, liposome membrane permeability, and changes in physicochemical characteristics of the liposomes. Fluorescence spectroscopy methods and Fourier transform infrared spectroscopy (ATR-FTIR), which provides information about specific groups in lipids involved in interaction with the protein, were used to monitor adsorption of albumin with liposomes based on egg phosphatidylcholine with various additives of negatively charged lipidic components, such as phosphatidylinositol, ganglioside GM1, or the acidic lipopeptide. Less than a dozen of the protein molecules were tightly bound to a liposome independently of bilayer composition, yet they had a detectable impact on the bilayer. Albumin conformational changes during adsorption were partially related to bilayer microhydrophobicity. Ganglioside GM1 showed preferable features for evading undesirable structural changes.
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- 2022
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5. Photorhabdus luminescens TccC3 Toxin Targets the Dynamic Population of F-Actin and Impairs Cell Cortex Integrity
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Songyu Dong, Weili Zheng, Nicholas Pinkerton, Jacob Hansen, Svetlana B. Tikunova, Jonathan P. Davis, Sarah M. Heissler, Elena Kudryashova, Edward H. Egelman, and Dmitri S. Kudryashov
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TccC3 ,actin ,ADP-ribosylation ,bacterial toxin ,calponin-homology domain ,actin dynamics ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Due to its essential role in cellular processes, actin is a common target for bacterial toxins. One such toxin, TccC3, is an effector domain of the ABC-toxin produced by entomopathogenic bacteria of Photorhabdus spp. Unlike other actin-targeting toxins, TccC3 uniquely ADP-ribosylates actin at Thr-148, resulting in the formation of actin aggregates and inhibition of phagocytosis. It has been shown that the fully modified F-actin is resistant to depolymerization by cofilin and gelsolin, but their effects on partially modified actin were not explored. We found that only F-actin unprotected by tropomyosin is the physiological TccC3 substrate. Yet, ADP-ribosylated G-actin can be produced upon cofilin-accelerated F-actin depolymerization, which was only mildly inhibited in partially modified actin. The affinity of TccC3-ADP-ribosylated G-actin for profilin and thymosin-β4 was weakened moderately but sufficiently to potentiate spontaneous polymerization in their presence. Interestingly, the Arp2/3-mediated nucleation was also potentiated by T148-ADP-ribosylation. Notably, even partially modified actin showed reduced bundling by plastins and α-actinin. In agreement with the role of these and other tandem calponin-homology domain actin organizers in the assembly of the cortical actin network, TccC3 induced intense membrane blebbing in cultured cells. Overall, our data suggest that TccC3 imposes a complex action on the cytoskeleton by affecting F-actin nucleation, recycling, and interaction with actin-binding proteins involved in the integration of actin filaments with each other and cellular elements.
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- 2022
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6. Plastin 3 in X-Linked Osteoporosis: Imbalance of Ca2+-Dependent Regulation Is Equivalent to Protein Loss
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Christopher L. Schwebach, Elena Kudryashova, and Dmitri S. Kudryashov
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plastin 3 ,X-linked osteoporosis ,osteogenesis imperfecta ,bone development ,actin bundling ,Ca2+-dependent regulation ,Biology (General) ,QH301-705.5 - Abstract
Osteogenesis imperfecta is a genetic disorder disrupting bone development and remodeling. The primary causes of osteogenesis imperfecta are pathogenic variants of collagen and collagen processing genes. However, recently variants of the actin bundling protein plastin 3 have been identified as another source of osteogenesis imperfecta. Plastin 3 is a highly conserved protein involved in several important cellular structures and processes and is controlled by intracellular Ca2+ which potently inhibits its actin-bundling activity. The precise mechanisms by which plastin 3 causes osteogenesis imperfecta remain unclear, but recent advances have contributed to our understanding of bone development and the actin cytoskeleton. Here, we review the link between plastin 3 and osteogenesis imperfecta highlighting in vitro studies and emphasizing the importance of Ca2+ regulation in the localization and functionality of plastin 3.
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- 2021
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7. Phosphatidylinositol Stabilizes Fluid-Phase Liposomes Loaded with a Melphalan Lipophilic Prodrug
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Daria Tretiakova, Irina Le-Deigen, Natalia Onishchenko, Judith Kuntsche, Elena Kudryashova, and Elena Vodovozova
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nanosized liposomes ,lipophilic prodrug ,melphalan ,phosphatidylinositol ,albumin ,ATR-FTIR spectroscopy ,Pharmacy and materia medica ,RS1-441 - Abstract
Previously, a liposomal formulation of a chemotherapeutic agent melphalan (Mlph) incorporated in a fluid lipid bilayer of natural phospholipids in the form of dioleoylglyceride ester (MlphDG) was developed and the antitumor effect was confirmed in mouse models. The formulation composed of egg phosphatidylcholine (ePC), soybean phosphatidylinositol (PI), and MlphDG (8:1:1, by mol) showed stability in human serum for at least 4–5 h. On the contrary, replacing PI with pegylation of the liposomes, promoted fast dissociation of the components from the bilayer. In this work, interactions of MlphDG-liposomes with the most abundant plasma protein—albumin—in function of the presence of PI in the formulation were explored using Fourier transform infrared spectroscopy. The release of MlphDG from the liposomes was studied by asymmetrical flow field-flow fractionation (AF4) using micelles formed by a polyethylene glycol conjugate with phosphatidylethanolamine to mimic the physiological lipid sink like lipoproteins. Our results show that PI actually protects the membrane of MlphDG-liposomes from the protein penetration, presumably due to pairing between the positively charged MlphDG and negatively charged PI, which compensates for the heterogeneity of the lipid bilayer. The AF4 technique also evidences high stability of the formulation as a drug carrier.
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- 2021
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8. Rounding Out the Understanding of ACD Toxicity with the Discovery of Cyclic Forms of Actin Oligomers
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Harper Smith, Nick Pinkerton, David B. Heisler, Elena Kudryashova, Aaron R. Hall, Kelly R. Karch, Andrew Norris, Vicki Wysocki, Marcos Sotomayor, Emil Reisler, Dimitrios Vavylonis, and Dmitri S. Kudryashov
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bacterial toxins ,actin ,actin cross-linking domain ,ACD ,MARTX ,isopeptide bond ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Actin is an essential element of both innate and adaptive immune systems and can aid in motility and translocation of bacterial pathogens, making it an attractive target for bacterial toxins. Pathogenic Vibrio and Aeromonas genera deliver actin cross-linking domain (ACD) toxin into the cytoplasm of the host cell to poison actin regulation and promptly induce cell rounding. At early stages of toxicity, ACD covalently cross-links actin monomers into oligomers (AOs) that bind through multivalent interactions and potently inhibit several families of actin assembly proteins. At advanced toxicity stages, we found that the terminal protomers of linear AOs can get linked together by ACD to produce cyclic AOs. When tested against formins and Ena/VASP, linear and cyclic AOs exhibit similar inhibitory potential, which for the cyclic AOs is reduced in the presence of profilin. In coarse-grained molecular dynamics simulations, profilin and WH2-motif binding sites on actin subunits remain exposed in modeled AOs of both geometries. We speculate, therefore, that the reduced toxicity of cyclic AOs is due to their reduced configurational entropy. A characteristic feature of cyclic AOs is that, in contrast to the linear forms, they cannot be straightened to form filaments (e.g., through stabilization by cofilin), which makes them less susceptible to neutralization by the host cell.
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- 2021
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9. Oligomerization Affects the Ability of Human Cyclase-Associated Proteins 1 and 2 to Promote Actin Severing by Cofilins
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Vedud Purde, Florian Busch, Elena Kudryashova, Vicki H. Wysocki, and Dmitri S. Kudryashov
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cyclase-associated proteins ,oligomerization ,coiled coils ,actin severing ,actin depolymerization ,α-barrels ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Actin-depolymerizing factor (ADF)/cofilins accelerate actin turnover by severing aged actin filaments and promoting the dissociation of actin subunits. In the cell, ADF/cofilins are assisted by other proteins, among which cyclase-associated proteins 1 and 2 (CAP1,2) are particularly important. The N-terminal half of CAP has been shown to promote actin filament dynamics by enhancing ADF-/cofilin-mediated actin severing, while the central and C-terminal domains are involved in recharging the depolymerized ADP−G-actin/cofilin complexes with ATP and profilin. We analyzed the ability of the N-terminal fragments of human CAP1 and CAP2 to assist human isoforms of “muscle” (CFL2) and “non-muscle” (CFL1) cofilins in accelerating actin dynamics. By conducting bulk actin depolymerization assays and monitoring single-filament severing by total internal reflection fluorescence (TIRF) microscopy, we found that the N-terminal domains of both isoforms enhanced cofilin-mediated severing and depolymerization at similar rates. According to our analytical sedimentation and native mass spectrometry data, the N-terminal recombinant fragments of both human CAP isoforms form tetramers. Replacement of the original oligomerization domain of CAPs with artificial coiled-coil sequences of known oligomerization patterns showed that the activity of the proteins is directly proportional to the stoichiometry of their oligomerization; i.e., tetramers and trimers are more potent than dimers, which are more effective than monomers. Along with higher binding affinities of the higher-order oligomers to actin, this observation suggests that the mechanism of actin severing and depolymerization involves simultaneous or consequent and coordinated binding of more than one N-CAP domain to F-actin/cofilin complexes.
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- 2019
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10. Glutamyl phosphate is an activated intermediate in actin crosslinking by actin crosslinking domain (ACD) toxin.
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Elena Kudryashova, Caitlin Kalda, and Dmitri S Kudryashov
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Medicine ,Science - Abstract
Actin Crosslinking Domain (ACD) is produced by several life-threatening Gram-negative pathogenic bacteria as part of larger toxins and delivered into the cytoplasm of eukaryotic host cells via Type I or Type VI secretion systems. Upon delivery, ACD disrupts the actin cytoskeleton by catalyzing intermolecular amide bond formation between E270 and K50 residues of actin, leading to the formation of polymerization-deficient actin oligomers. Ultimately, accumulation of the crosslinked oligomers results in structural and functional failure of the actin cytoskeleton in affected cells. In the present work, we advanced in our understanding of the ACD catalytic mechanism by discovering that the enzyme transfers the gamma-phosphoryl group of ATP to the E270 actin residue, resulting in the formation of an activated acyl phosphate intermediate. This intermediate is further hydrolyzed and the energy of hydrolysis is utilized for the formation of the amide bond between actin subunits. We also determined the pH optimum for the reaction and the kinetic parameters of ACD catalysis for its substrates, ATP and actin. ACD showed sigmoidal, non-Michaelis-Menten kinetics for actin (K(0.5) = 30 µM) reflecting involvement of two actin molecules in a single crosslinking event. We established that ACD can also utilize Mg(2+)-GTP to support crosslinking, but the kinetic parameters (K(M) = 8 µM and 50 µM for ATP and GTP, respectively) suggest that ATP is the primary substrate of ACD in vivo. The optimal pH for ACD activity was in the range of 7.0-9.0. The elucidated kinetic mechanism of ACD toxicity adds to understanding of complex network of host-pathogen interactions.
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- 2012
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11. Discrete Phase-Locked Loop Systems and Spreadsheets
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Sergei Abramovich, Elena Kudryashova, Gennady A Leonov, and Stephen J. Sugden
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phase locked loops ,clock skew ,difference equations ,spreadsheet modeling ,orbit diagrams ,period-doubling bifurcations ,tertiary education ,Special aspects of education ,LC8-6691 - Abstract
This paper demonstrates the use of a spreadsheet in exploring non-linear difference equations that describe digital control systems used in radio engineering, communication and computer architecture. These systems, being the focus of intensive studies of mathematicians and engineers over the last 40 years, may exhibit extremely complicated behavior interpreted in contemporary terms as transition from global asymptotic stability to chaos through period-doubling bifurcations. The authors argue that embedding advanced mathematical ideas in the technological tool enables one to introduce fundamentals of discrete control systems in tertiary curricula without learners having to deal with complex machinery that rigorous mathematical methods of investigation require. In particular, in the appropriately designed spreadsheet environment, one can effectively visualize a qualitative difference in the behavior of systems with different types of non-linear characteristic.
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- 2005
12. Computer simulation of the Receptor–Ligand Interactions of Mannose Receptor CD206 in Comparison with the Lectin Concanavalin A Model
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Elena Kudryashova and Igor Zlotnikov
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Carbohydrates ,General Medicine ,Molecular Dynamics Simulation ,Ligands ,Biochemistry ,Article ,molecular dynamics ,macrophages ,CD206 ,Lectins ,Humans ,Mannose Receptor ,artificial neural network ,concanavalin A - Abstract
Computer modeling of complexation of mono- and oligosaccharide ligands with the main (fourth) carbohydrate-binding domain of the mannose receptor CD206 (CRD4), as well as with the model receptor concanavalin A (ConA), was carried out for the first time, using methods of molecular dynamics and neural network analysis. ConA was shown to be a relevant model of CD206 (CRD4) due to similarity of the structural organization of the binding sites and high correlation of the values of free energies of complexation between the literature data and computer modeling (r > 0.9). Role of the main factors affecting affinity of the ligand–receptor interactions is discussed: the number and nature of carbohydrate residues, presence of Me-group in the O1 position, type of the glycoside bond in dimannose. Complexation of ConA and CD206 with ligands is shown to be energetically caused by electrostatic interactions (E) of the charged residues (Asn, Asp, Arg) with oxygen and hydrogen atoms in carbohydrates; contributions of hydrophobic and van der Waals components is lower. Possibility of the additional stabilization of complexes due to the CH–π stacking interactions of Tyr with the Man plane is discussed. The role of calcium and manganese ions in binding ligands has been studied. The values of free energies of complexation calculated in the course of molecular dynamics simulation correlate with experimental data (published for the model ConA): correlation coefficient r = 0.68. The Pafnucy neural network was trained based on the set of PDBbind2020 ligand–receptor complexes, which significantly increased accuracy of the energy predictions to r = 0.8 and 0.82 for CD206 and ConA receptors, respectively. A model of normalization of the complexation energy values for calculating the relevant values of ΔGbind, Kd is proposed. Based on the developed technique, values of the dissociation constants of a series of CD206 complexes with nine carbohydrate ligands of different structures were determined, which were not previously known. The obtained data open up possibilities for using computer modeling for the development of optimal drug carriers capable of active macrophage targeting, and also determine the limits of applicability of using ConA as a relevant model for studying parameters of the CD206 binding to various carbohydrate ligands. Electronic supplementary material The online version contains supplementary material available at 10.1134/S0006297922010059.
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- 2022
13. Pointed-end processive elongation of actin filaments by
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Elena, Kudryashova, Ankita, Heidi, Ulrichs, Shashank, Shekhar, and Dmitri S, Kudryashov
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According to the cellular actin dynamics paradigm, filaments grow at their barbed ends and depolymerize predominantly from their pointed ends to form polar structures and do productive work. We show that actin can elongate at the pointed end when assisted by
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- 2022
14. The effect of antifibrotic and antibacterial drugs on the physicochemical properties of liposomes: A spectral study
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Irina Le-Deygen, Anastasia Safronova, Polina Mamaeva, Anna Skuredina, and Elena Kudryashova
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- 2022
15. Intein-mediated cytoplasmic reconstitution of a split toxin enables selective cell ablation in mixed populations and tumor xenografts
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Elena Kudryashova, Reena Shakya, Dmitri S. Kudryashov, David B. Heisler, and Vedud Purde
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0301 basic medicine ,Cytoplasm ,Bacterial Toxins ,Cell ,Mice, Nude ,Receptors, Cell Surface ,010402 general chemistry ,01 natural sciences ,Inteins ,Mice ,03 medical and health sciences ,Drug Delivery Systems ,Protein Domains ,Immunotoxin ,Cell Line, Tumor ,Neoplasms ,medicine ,Animals ,Humans ,Diphtheria Toxin ,Receptor ,Diphtheria toxin ,Multidisciplinary ,Chemistry ,Immunotoxins ,Biological Sciences ,Transmembrane protein ,0104 chemical sciences ,Cell biology ,Protein Transport ,030104 developmental biology ,medicine.anatomical_structure ,Cancer cell ,Heterografts ,Female ,Intein - Abstract
The application of proteinaceous toxins for cell ablation is limited by their high on- and off-target toxicity, severe side effects, and a narrow therapeutic window. The selectivity of targeting can be improved by intein-based toxin reconstitution from two dysfunctional fragments provided their cytoplasmic delivery via independent, selective pathways. While the reconstitution of proteins from genetically encoded elements has been explored, exploiting cell-surface receptors for boosting selectivity has not been attained. We designed a robust splitting algorithm and achieved reliable cytoplasmic reconstitution of functional diphtheria toxin from engineered intein-flanked fragments upon receptor-mediated delivery of one of them to the cells expressing the counterpart. Retargeting the delivery machinery toward different receptors overexpressed in cancer cells enables selective ablation of specific subpopulations in mixed cell cultures. In a mouse model, the transmembrane delivery of a split-toxin construct potently inhibits the growth of xenograft tumors expressing the split counterpart. Receptor-mediated delivery of engineered split proteins provides a platform for precise therapeutic and experimental ablation of tumors or desired cell populations while also greatly expanding the applicability of the intein-based protein transsplicing.
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- 2020
16. Involvement of Smi1 in cell wall integrity and glucan synthase Bgs4 localization during fission yeast cytokinesis
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Larissa V. G. Longo, Evelyn G. Goodyear, Sha Zhang, Elena Kudryashova, and Jian-Qiu Wu
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Saccharomyces cerevisiae Proteins ,beta-Glucans ,Cell Membrane ,Membrane Proteins ,Cell Biology ,Actomyosin ,Actin Cytoskeleton ,Cell Wall ,Glucosyltransferases ,Schizosaccharomyces ,Schizosaccharomyces pombe Proteins ,Molecular Biology ,Cytokinesis ,Transcription Factors - Abstract
Cytokinesis is the final step of the cell-division cycle. In fungi, it relies on the coordination of constriction of an actomyosin contractile ring and construction of the septum at the division site. Glucan synthases synthesize glucans, which are the major components in fungal cell walls and division septa. It is known that Rho1 and Rho2 GTPases regulate glucan synthases Bgs1, Bgs4, and Ags1, and that Sbg1 and the F-BAR protein Cdc15 play roles in Bgs1 stability and delivery to the plasma membrane. Here we characterize Smi1, an intrinsically disordered protein that interacts with Bgs4 and regulates its trafficking and localization in fission yeast. Smi1 is important for septum integrity, and its absence causes severe lysis during cytokinesis. Smi1 localizes to secretory vesicles and moves together with Bgs4 toward the division site. The concentrations of the glucan synthases Bgs1 and Bgs4 and the glucanases Agn1 and Bgl2 decrease at the division site in the
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- 2021
17. SARS-CoV-2 inactivation by human defensin HNP1 and retrocyclin RC-101
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Wuyuan Lu, Ashley Zani, Elena Kudryashova, Dmitri S. Kudryashov, Amit Sharma, Jacob S. Yount, and Geraldine Vilmen
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Vaccination ,Innate immune system ,Immune system ,Viral envelope ,biology ,viruses ,fungi ,Serum albumin ,biology.protein ,Lipid bilayer fusion ,Virology ,Defensin ,Virus - Abstract
Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is an enveloped virus responsible for the COVID-19 respiratory disease pandemic. While induction of adaptive antiviral immunity via vaccination holds promise for combatting the pandemic, the emergence of new potentially more transmissible and vaccine-resistant variants of SARS-CoV-2 is an ever-present threat. Thus, it remains essential to better understand innate immune mechanisms that are active against the virus. One component of the innate immune system with broad anti-pathogen, including antiviral, activity is a group of cationic immune peptides termed defensins. The defensins’ ability to neutralize enveloped and non-enveloped viruses and to inactivate numerous bacterial toxins correlate with their ability to promote the unfolding of thermodynamically pliable proteins. Accordingly, we found that human neutrophil a-defensin HNP1 and retrocyclin RC-101 destabilize SARS-CoV-2 Spike protein and interfere with Spike-mediated membrane fusion and SARS-CoV-2 infection in cell culture. We show that HNP1 binds to Spike with submicromolar affinity. Although binding of HNP1 to serum albumin is more than 20-fold weaker, serum reduces the anti-SARS-CoV-2 activity of HNP1. At high concentrations of HNP1, its ability to inactivate the virus was preserved even in the presence of serum. These results suggest that specific a- and 8-defensins may be valuable tools in developing SARS-CoV-2 infection prevention strategies.
- Published
- 2021
18. Cross-border Cooperation Programmes as a Resource of Social and Economic Development of Barents Region (Case of ENPI Kolarctic Programme)
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Lyubov’ Zarubina and Elena Kudryashova
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Economics and Econometrics ,Economic growth ,Resource (biology) ,Political Science and International Relations ,Business ,Cross-border cooperation - Published
- 2019
19. Scenarios for the development of the Arctic region (2020–2035)
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Nikolay A. Kondratov, Svetlana Lipina, Anatoliy Chistobaev, Elena Kudryashova, and Konstantin Zaikov
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socio-ecological systems ,climate change ,Oceanography ,Political science ,Social Sciences ,geopolitics ,General Medicine ,development strategies ,the arctic ,innovation ,The arctic - Abstract
The importance of selecting the development of the Arctic seems to be relevant since rapid and irreversible changes are taking place there. Climate change and globalization are their prominent examples. A complex of factors has both positive and negative impacts on the use of natural resources and the positioning of states located not only within the Arctic but also outside it. The questions arise: what is the significance of these changes for geography, politics, and the management system? How should the comprehension of these processes be built? The relevance of the topic is enhanced by the fact that Russia has the most significant Arctic sector among the states with access to the Arctic Ocean. Therefore, our country has a leading role in working out strategies for the development of the Arctic. The comprehensive approach (considering the economic and political-geographical positions) is central in the article to analyze the directions of development of the Arctic territories. The method reveals the possibilities of sustainable development, which will provide Russia with strategic benefits within the Arctic and globally. The article discusses scenarios for the development of the Arctic, including the Arctic zone of the Russian Federation, in the long-term perspective (until 2035). Substantiation of the long-term prospects for the development of the Arctic, despite Russian and foreign research, seems to be unrealistic due to lack of knowledge about the nature and consequences of climatic changes currently observed in this region and affecting global environmental management. The authors concluded that the priority directions of the Arctic development should be the ones based on positive and innovative trends.
- Published
- 2019
20. Cross-Border Investment Cooperation in the Arctic Region: Challenges and Opportunities
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Irina Sivobrova, Elena Kudryashova, and Lyubov’ Zarubina
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arctic region ,lcsh:Sociology (General) ,investment attractiveness strategy ,kolarctic cooperation program ,lcsh:HM401-1281 ,Business ,International economics ,Investment (macroeconomics) ,cross-border investment cooperation ,best investment practices ,The arctic ,large-scale infrastructure projects - Abstract
The article considers the investment processes that take place in the foreign and Russian Arctic, the tools to attract investment and the possibility of accumulating best practices in the implementation of investment projects. Analysis of the data on planned, unfinished and cancelled projects in the Russian and foreign Arctic, including joint projects, allows us to reveal problems in cross-border investment cooperation. In the framework of global economic challenges, when discussing the opportunities for the Northern regions alternative to large-scale production and transportation, we determine the need to find new promising areas of investment, including infrastructure projects. On the basis of official, statistical and expert assessments, we analyze the priorities and directions of investment activities in the Arctic and the main tools to increase investment attractiveness and promote investment. We consider European Neighbourhood and Partnership Instrument (ENPI) Kolarctic 2007–2013 as an analytical case, since it is one of the most effective tools for providing financial support to multilateral cross-border cooperation projects in the Barents Euro-Arctic Region. The project field of the program is quite extensive and contains a variety of topics; thsi contributed to the promotion of cross-border cooperation in various fields, involvement of different target groups and stakeholders, and development of project cooperation between different sectors in the Barents Euro-Arctic Region. We find it necessary to consider foreign practice of the comprehensive use of both direct and indirect methods for attracting investments; on its basis it can be possible to create the banks of best investment practices in Russia’s Northern regions. Having revealed the inertia of investment processes in the Arctic, we conclude that it is necessary to develop a strategy to increase the investment attractiveness of the Northern territories
- Published
- 2019
21. The Need for Workforce in Constituent Entities of the Arctic Zone of the Russian Federation
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Aleksandr Tamitskii, Nikolai Kondratov, Elena Kudryashova, and Konstantin Zaikov
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education ,workforce ,05 social sciences ,lcsh:HM401-1281 ,010501 environmental sciences ,development strategy ,arctic zone of russia ,01 natural sciences ,need for personnel ,The arctic ,lcsh:Sociology (General) ,Economy ,Political science ,0502 economics and business ,Workforce ,Russian federation ,050203 business & management ,0105 earth and related environmental sciences - Abstract
In the first quarter of the 21st century, the Arctic and everything connected with it remains a popular topic in the humanitarian, socio-economic and political spheres. It is especially relevant for Russia, which has the largest polar sector in the world. An integrated approach to the study and analysis of development prospects of the Arctic territories can create conditions for sustainable socio-economic development not only in the Arctic zone of Russia, but also in Russia as a whole. One of the elements of this approach is the need for skilled workforce at the enterprises of the Arctic zone of the Russian Federation; this topic is considered in the paper. The relevance of the topic stems from the need to implement large-scale investment projects for development of mineral resources and transport infrastructure (megaprojects), the need to address long-term tasks of socio-economic development of the Russian Arctic and ensure national security in the region. All these points are contained in the strategy for development of the Arctic zone of the Russian Federation until 2020. Statistical evaluation of demographic processes indicates a continuous outflow of population from the majority of subjects of the Arctic zone of Russia. This happens both due to natural reasons and as a result of emigration, including the emigration of working age individuals. Scientific novelty of the paper consists in its comprehensive approach: using analytical and sociological methods, we analyze the need for labor force in the real sector of the economy in the Russian Arctic. We consider the possibilities of the higher education system to meet such needs. It is established that the emigration of able-bodied population from the subjects of the Russian Arctic and the launch of megaprojects can be considered as factors in the formation of the need for labor resources in the subjects of the Arctic zone of Russia. Further scientific search in the chosen topic consists in the following points: development of methods for long-term forecasting of the needs of economic entities in labor force against the background of its outflow from the Arctic zone, additional substantiation of the medium-term need and the possibility of its implementation through higher education, and search for an alternative to corporate data on the need for workforce
- Published
- 2019
22. International Projects as a Tool of Regional Development and Cooperation Enhancement (Case of Arkhangelsk Region Participation in Kolarctic CBC Programme)
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Lyubov’ Zarubina and Elena Kudryashova
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Regional development ,Political science ,Regional science - Published
- 2018
23. Plastin 3 in X-Linked Osteoporosis: Imbalance of Ca2+-Dependent Regulation Is Equivalent to Protein Loss
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Elena Kudryashova, Christopher L. Schwebach, and Dmitri S. Kudryashov
- Subjects
Mini Review ,Osteoporosis ,X-linked osteoporosis ,osteogenesis imperfecta ,Biology ,Cell and Developmental Biology ,actin bundling ,medicine ,PLS3 ,lcsh:QH301-705.5 ,Gene ,plastin 3 ,Genetic disorder ,Cell Biology ,Ca2+-dependent regulation ,medicine.disease ,Actin cytoskeleton ,In vitro ,T-Plastin ,Cell biology ,lcsh:Biology (General) ,Osteogenesis imperfecta ,bone development ,Intracellular ,Developmental Biology - Abstract
Osteogenesis imperfecta is a genetic disorder disrupting bone development and remodeling. The primary causes of osteogenesis imperfecta are pathogenic variants of collagen and collagen processing genes. However, recently variants of the actin bundling protein plastin 3 have been identified as another source of osteogenesis imperfecta. Plastin 3 is a highly conserved protein involved in several important cellular structures and processes and is controlled by intracellular Ca2+which potently inhibits its actin-bundling activity. The precise mechanisms by which plastin 3 causes osteogenesis imperfecta remain unclear, but recent advances have contributed to our understanding of bone development and the actin cytoskeleton. Here, we review the link between plastin 3 and osteogenesis imperfecta highlightingin vitrostudies and emphasizing the importance of Ca2+regulation in the localization and functionality of plastin 3.
- Published
- 2021
24. Rounding Out the Understanding of ACD Toxicity with the Discovery of Cyclic Forms of Actin Oligomers
- Author
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Dimitrios Vavylonis, Dmitri S. Kudryashov, Vicki H. Wysocki, Marcos Sotomayor, Harper Smith, David B. Heisler, Elena Kudryashova, Emil Reisler, Aaron R. Hall, Andrew Norris, Kelly R Karch, and Nick Pinkerton
- Subjects
0301 basic medicine ,isopeptide bond ,formin ,Cell ,macromolecular substances ,Catalysis ,Article ,Inorganic Chemistry ,lcsh:Chemistry ,03 medical and health sciences ,0302 clinical medicine ,MARTX ,medicine ,multivalent interactions ,Physical and Theoretical Chemistry ,Binding site ,Ena/VASP ,Molecular Biology ,lcsh:QH301-705.5 ,ACD ,Spectroscopy ,Actin ,bacterial toxins ,mass spectrometry ,chemistry.chemical_classification ,Isopeptide bond ,biology ,Chemistry ,actin cross-linking domain ,Organic Chemistry ,General Medicine ,Cofilin ,molecular dynamics ,Computer Science Applications ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Profilin ,lcsh:Biology (General) ,lcsh:QD1-999 ,Cytoplasm ,Formins ,polymerization ,biology.protein ,actin ,030217 neurology & neurosurgery - Abstract
Actin is an essential element of both innate and adaptive immune systems and can aid in motility and translocation of bacterial pathogens, making it an attractive target for bacterial toxins. Pathogenic Vibrio and Aeromonas genera deliver actin cross-linking domain (ACD) toxin into the cytoplasm of the host cell to poison actin regulation and promptly induce cell rounding. At early stages of toxicity, ACD covalently cross-links actin monomers into oligomers (AOs) that bind through multivalent interactions and potently inhibit several families of actin assembly proteins. At advanced toxicity stages, we found that the terminal protomers of linear AOs can get linked together by ACD to produce cyclic AOs. When tested against formins and Ena/VASP, linear and cyclic AOs exhibit similar inhibitory potential, which for the cyclic AOs is reduced in the presence of profilin. In coarse-grained molecular dynamics simulations, profilin and WH2-motif binding sites on actin subunits remain exposed in modeled AOs of both geometries. We speculate, therefore, that the reduced toxicity of cyclic AOs is due to their reduced configurational entropy. A characteristic feature of cyclic AOs is that, in contrast to the linear forms, they cannot be straightened to form filaments (e.g., through stabilization by cofilin), which makes them less susceptible to neutralization by the host cell.
- Published
- 2021
25. Opposing activities of IFITM proteins in SARS-CoV-2 infection
- Author
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Guoli Shi, Kin Kui Lai, Alex A. Compton, Luanne Hall-Stoodley, Ashley Zani, Elena Kudryashova, Adam D. Kenney, Lizhi Zhang, Richard T. Robinson, Jacob S. Yount, and Dmitri S. Kudryashov
- Subjects
Endosome ,viruses ,Endocytic cycle ,Mutant ,Biology ,Article ,SARS‐CoV‐2 ,General Biochemistry, Genetics and Molecular Biology ,Virus ,Cell Line ,IFITM ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Palmitoylation ,COVID‐19 ,Interferon ,Chlorocebus aethiops ,Plasma membrane fusion ,medicine ,Animals ,Humans ,Enhancer ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,General Immunology and Microbiology ,SARS-CoV-2 ,General Neuroscience ,Serine Endopeptidases ,COVID-19 ,Membrane Proteins ,RNA-Binding Proteins ,Articles ,Virus Internalization ,Antigens, Differentiation ,Microbiology, Virology & Host Pathogen Interaction ,Transmembrane protein ,Cell biology ,IFITM3 ,Membrane protein ,Mutation ,Angiotensin-Converting Enzyme 2 ,030217 neurology & neurosurgery ,medicine.drug - Abstract
Interferon-induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. Here we show that SARS-CoV-2 Spike-pseudotyped virus and genuine SARS-CoV-2 infections are generally restricted by expression of human IFITM1, IFITM2, and IFITM3, using both gain- and loss-of-function approaches. Mechanistically, restriction of SARS-CoV-2 occurred independently of IFITM3S-palmitoylation sites, indicating a restrictive capacity that is distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the human IFITM3 endocytosis-promoting YxxΦ motif converted human IFITM3 into an enhancer of SARS-CoV-2 infection, and cell-to-cell fusion assays confirmed the ability of endocytic mutants to enhance Spike-mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which reportedly increases plasma membrane fusion versus endosome fusion of SARS-CoV-2, attenuated IFITM3 restriction and converted amphipathic helix mutants into strong enhancers of infection. In sum, these data uncover new pro- and anti-viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity-based mechanisms used for endosomal virus restriction. Indeed, the net effect of IFITM3 on SARS-CoV-2 infections may be a result of these opposing activities, suggesting that shifts in the balance of these activities could be coopted by viruses to escape this important first line innate defense mechanism.
- Published
- 2020
26. Oligomerization Affects the Ability of Human Cyclase-Associated Proteins 1 and 2 to Promote Actin Severing by Cofilins
- Author
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Elena Kudryashova, Vicki H. Wysocki, Dmitri S. Kudryashov, Vedud Purde, and Florian Busch
- Subjects
0301 basic medicine ,α-barrels ,Protein Conformation ,actin depolymerization ,Sequence Homology ,Cell Cycle Proteins ,actin severing ,lcsh:Chemistry ,Protein filament ,0302 clinical medicine ,Protein Isoforms ,lcsh:QH301-705.5 ,Spectroscopy ,biology ,Chemistry ,cyclase-associated proteins ,General Medicine ,Cofilin ,coiled coils ,3. Good health ,Computer Science Applications ,Actin Depolymerizing Factors ,Profilin ,030220 oncology & carcinogenesis ,Gene isoform ,macromolecular substances ,Cyclase ,Article ,Catalysis ,oligomerization ,Inorganic Chemistry ,03 medical and health sciences ,Humans ,Amino Acid Sequence ,Physical and Theoretical Chemistry ,Molecular Biology ,Actin ,Adaptor Proteins, Signal Transducing ,Total internal reflection fluorescence microscope ,Depolymerization ,Organic Chemistry ,Membrane Proteins ,Actins ,Cytoskeletal Proteins ,030104 developmental biology ,lcsh:Biology (General) ,lcsh:QD1-999 ,Biophysics ,biology.protein ,Protein Multimerization - Abstract
Actin-depolymerizing factor (ADF)/cofilins accelerate actin turnover by severing aged actin filaments and promoting the dissociation of actin subunits. In the cell, ADF/cofilins are assisted by other proteins, among which cyclase-associated proteins 1 and 2 (CAP1,2) are particularly important. The N-terminal half of CAP has been shown to promote actin filament dynamics by enhancing ADF-/cofilin-mediated actin severing, while the central and C-terminal domains are involved in recharging the depolymerized ADP&ndash, G-actin/cofilin complexes with ATP and profilin. We analyzed the ability of the N-terminal fragments of human CAP1 and CAP2 to assist human isoforms of &ldquo, muscle&rdquo, (CFL2) and &ldquo, non-muscle&rdquo, (CFL1) cofilins in accelerating actin dynamics. By conducting bulk actin depolymerization assays and monitoring single-filament severing by total internal reflection fluorescence (TIRF) microscopy, we found that the N-terminal domains of both isoforms enhanced cofilin-mediated severing and depolymerization at similar rates. According to our analytical sedimentation and native mass spectrometry data, the N-terminal recombinant fragments of both human CAP isoforms form tetramers. Replacement of the original oligomerization domain of CAPs with artificial coiled-coil sequences of known oligomerization patterns showed that the activity of the proteins is directly proportional to the stoichiometry of their oligomerization, i.e., tetramers and trimers are more potent than dimers, which are more effective than monomers. Along with higher binding affinities of the higher-order oligomers to actin, this observation suggests that the mechanism of actin severing and depolymerization involves simultaneous or consequent and coordinated binding of more than one N-CAP domain to F-actin/cofilin complexes.
- Published
- 2019
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27. Creation of development zones in the Arctic: methodology and practice
- Author
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Svetlana Lipina, Yulia Bogdanova, Olga Smirnova, Tatyana Krejdenko, and Elena Kudryashova
- Subjects
Engineering ,supporting areas ,business.industry ,Environmental resource management ,regional economics ,the development of the Northern Sea Route ,Social Sciences ,General Medicine ,strategic planning ,the NSR ,The arctic ,Arctic ,spatial development ,national security ,business ,the government program - Abstract
The article describes the basic principles and methodological bases of formation and development of the supporting areas of the Russian Arctic. The work emphasizes that the basis of the methodological approach in the formation of the supporting areas is a vector of development of the territory as an integral project on the principle of coordination of all "industrial" activities in the planning, goal-setting, financing and implementation, which will allow to reduce all kinds of costs and expenses, as well as all the projects included in the supporting areas should be aimed at the development of the macro-region as a whole, not just to solve individual tasks of the industry. The article underlines that the formation of the supporting areas is aimed at achieving a single global goal — to encourage efficiency and diversification of the economy of the Arctic zone, oriented to the preservation and development of the Northern Sea Route.
- Published
- 2016
28. Osteogenesis imperfecta mutations in plastin 3 lead to impaired calcium regulation of actin bundling
- Author
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Harper Smith, Elena Kudryashova, Matthew Orchard, Edward H. Egelman, Dmitri S. Kudryashov, Christopher L. Schwebach, Weili Zheng, and Lucas A. Runyan
- Subjects
0301 basic medicine ,Histology ,Physiology ,Endocrinology, Diabetes and Metabolism ,Mutant ,macromolecular substances ,Pathogenesis ,lcsh:Physiology ,Article ,Focal adhesion ,03 medical and health sciences ,0302 clinical medicine ,medicine ,PLS3 ,Bone ,lcsh:QH301-705.5 ,Calcium metabolism ,lcsh:QP1-981 ,Chemistry ,medicine.disease ,Actin bundling ,Cell biology ,030104 developmental biology ,lcsh:Biology (General) ,Osteogenesis imperfecta ,Lamellipodium ,030217 neurology & neurosurgery - Abstract
Mutations in actin-bundling protein plastin 3 (PLS3) emerged as a cause of congenital osteoporosis, but neither the role of PLS3 in bone development nor the mechanisms underlying PLS3-dependent osteoporosis are understood. Of the over 20 identified osteoporosis-linked PLS3 mutations, we investigated all five that are expected to produce full-length protein. One of the mutations distorted an actin-binding loop in the second actin-binding domain of PLS3 and abolished F-actin bundling as revealed by cryo-EM reconstruction and protein interaction assays. Surprisingly, the remaining four mutants fully retained F-actin bundling ability. However, they displayed defects in Ca2+ sensitivity: two of the mutants lost the ability to be inhibited by Ca2+, while the other two became hypersensitive to Ca2+. Each group of the mutants with similar biochemical properties showed highly characteristic cellular behavior. Wild-type PLS3 was distributed between lamellipodia and focal adhesions. In striking contrast, the Ca2+-hyposensitive mutants were not found at the leading edge but localized exclusively at focal adhesions/stress fibers, which displayed reinforced morphology. Consistently, the Ca2+-hypersensitive PLS3 mutants were restricted to lamellipodia, while chelation of Ca2+ caused their redistribution to focal adhesions. Finally, the bundling-deficient mutant failed to co-localize with any F-actin structures in cells despite a preserved F-actin binding through a non-mutation-bearing actin-binding domain. Our findings revealed that severe osteoporosis can be caused by a mutational disruption of the Ca2+-controlled PLS3’s cycling between adhesion complexes and the leading edge. Integration of the structural, biochemical, and cell biology insights enabled us to propose a molecular mechanism of plastin activity regulation by Ca2+.
- Published
- 2019
29. Actin Cross-Linking Toxin Is a Universal Inhibitor of Tandem-Organized and Oligomeric G-Actin Binding Proteins
- Author
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Elena Kudryashova, Alyssa J Harker, Dimitrios Vavylonis, David R. Kovar, David B. Heisler, Kyle Shafer, Dmitri S. Kudryashov, Margot E. Quinlan, and Blake Williams
- Subjects
0301 basic medicine ,actin cytoskeleton ,nucleation promoting factors ,Amino Acid Motifs ,Bacterial Toxins ,macromolecular substances ,medicine.disease_cause ,DNA-binding protein ,multivalent interaction ,Medical and Health Sciences ,General Biochemistry, Genetics and Molecular Biology ,WH2-domain ,Actin-Related Protein 2-3 Complex ,Article ,03 medical and health sciences ,0302 clinical medicine ,toxicity amplification ,Rare Diseases ,medicine ,actin-binding proteins ,Actin-binding protein ,Ena/VASP ,Vibrio cholerae ,Actin ,Total internal reflection fluorescence microscope ,biology ,Toxin ,Psychology and Cognitive Sciences ,Microfilament Proteins ,Biological Sciences ,Actin cytoskeleton ,Actins ,Cell biology ,single-molecule speckle live-cell microscopy ,Actin Cytoskeleton ,030104 developmental biology ,Orphan Drug ,Emerging Infectious Diseases ,Infectious Diseases ,Covalent bond ,Formins ,biology.protein ,Generic health relevance ,General Agricultural and Biological Sciences ,030217 neurology & neurosurgery ,cross-linking ,Developmental Biology - Abstract
Summary Delivery of bacterial toxins to host cells is hindered by host protective barriers. This obstruction dictates a remarkable efficiency of toxins, a single copy of which may kill a host cell. Efficiency of actin-targeting toxins is further hampered by an overwhelming abundance of their target. The actin cross-linking domain (ACD) toxins of Vibrio species and related bacterial genera catalyze the formation of covalently cross-linked actin oligomers. Recently, we reported that the ACD toxicity can be amplified via a multivalent inhibitory association of actin oligomers with actin assembly factors formins, suggesting that the oligomers may act as secondary toxins. Importantly, many proteins involved in nucleation, elongation, severing, branching, and bundling of actin filaments contain G-actin-binding Wiskott-Aldrich syndrome protein (WASP)-homology motifs 2 (WH2) organized in tandem and therefore may act as a multivalent platform for high-affinity interaction with the ACD-cross-linked actin oligomers. Using live-cell single-molecule speckle (SiMS) microscopy, total internal reflection fluorescence (TIRF) microscopy, and actin polymerization assays, we show that, in addition to formins, the oligomers bind with high affinity and potently inhibit several families of actin assembly factors: Ena/vasodilator-stimulated phosphorprotein (VASP); Spire; and the Arp2/3 complex, both in vitro and in live cells. As a result, ACD blocks the actin retrograde flow and membrane dynamics and disrupts association of Ena/VASP with adhesion complexes. This study defines ACD as a universal inhibitor of tandem-organized G-actin binding proteins that overcomes the abundance of actin by redirecting the toxicity cascade toward less abundant targets and thus leading to profound disorganization of the actin cytoskeleton and disruption of actin-dependent cellular functions.
- Published
- 2017
30. ACD toxin–produced actin oligomers poison formin-controlled actin polymerization
- Author
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Elena Kudryashova, Cristian Suarez, Dimitrios Vavylonis, Konstantin G. Birukov, Narasimham L. Parinandi, David R. Kovar, Sainath R. Kotha, Jonathan D. Winkelman, Dmitri S. Kudryashov, David B. Heisler, and Dmitry O. Grinevich
- Subjects
Multidisciplinary ,biology ,Toxin ,macromolecular substances ,Microfilament Protein ,medicine.disease_cause ,Cell biology ,Intestinal mucosa ,Biochemistry ,Cytoplasm ,Vibrio cholerae ,Formins ,biology.protein ,medicine ,Cytoskeleton ,Actin - Abstract
A little toxin can do a lot The actin cross-linking domain (ACD) is an actin-specific toxin produced by several bacterial pathogens. Heisler et al. discovered that ACD's pathogenic mechanism involves a highly unusual toxicity amplification cascade. Rather than directly inactivating the actin cytoskeleton, ACD blocks the activity of formins, actin regulatory proteins that play crucial roles in numerous cellular activities. ACD is exceptionally potent, even though its substrate is the most abundant protein of a eukaryotic cell: actin. Science , this issue p. 535
- Published
- 2015
31. Human Defensins Facilitate Local Unfolding of Thermodynamically Unstable Regions of Bacterial Protein Toxins
- Author
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Elena Kudryashova, Wuyuan Lu, Vicki H. Wysocki, Dmitri S. Kudryashov, Royston S. Quintyn, and Stephanie Seveau
- Subjects
alpha-Defensins ,beta-Defensins ,Protein Conformation ,Proteolysis ,Bacterial Toxins ,Immunology ,Thermolysin ,Exotoxins ,Plasma protein binding ,Enterotoxin ,Cell Line ,Enterotoxins ,Protein structure ,Bacterial Proteins ,medicine ,Chymotrypsin ,Humans ,Immunology and Allergy ,Protein Unfolding ,chemistry.chemical_classification ,Antigens, Bacterial ,integumentary system ,biology ,medicine.diagnostic_test ,fungi ,hemic and immune systems ,respiratory system ,bacterial infections and mycoses ,Repressor Proteins ,Infectious Diseases ,Enzyme ,Beta defensin ,chemistry ,Biochemistry ,biology.protein ,Unfolded protein response ,Protein Binding - Abstract
SummaryDefensins are short cationic, amphiphilic, cysteine-rich peptides that constitute the front-line immune defense against various pathogens. In addition to exerting direct antibacterial activities, defensins inactivate several classes of unrelated bacterial exotoxins. To date, no coherent mechanism has been proposed to explain defensins’ enigmatic efficiency toward various toxins. In this study, we showed that binding of neutrophil α-defensin HNP1 to affected bacterial toxins caused their local unfolding, potentiated their thermal melting and precipitation, exposed new regions for proteolysis, and increased susceptibility to collisional quenchers without causing similar effects on tested mammalian structural and enzymatic proteins. Enteric α-defensin HD5 and β-defensin hBD2 shared similar toxin-unfolding effects with HNP1, albeit to different degrees. We propose that protein susceptibility to inactivation by defensins is contingent to their thermolability and conformational plasticity and that defensin-induced unfolding is a key element in the general mechanism of toxin inactivation by human defensins.
- Published
- 2014
32. Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy
- Author
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Emil Reisler, Tatyana Polenova, Dmitri S. Kudryashov, Elena Kudryashova, and Jenna Yehl
- Subjects
Models, Molecular ,0301 basic medicine ,Cofilin 2 ,Protein Conformation ,Nuclear Magnetic Resonance ,1.1 Normal biological development and functioning ,Arp2/3 complex ,macromolecular substances ,Microfilament ,Filamentous actin ,Article ,03 medical and health sciences ,Adenosine Triphosphate ,Models ,Underpinning research ,Humans ,Actin-binding protein ,Nuclear Magnetic Resonance, Biomolecular ,Binding Sites ,Multidisciplinary ,030102 biochemistry & molecular biology ,biology ,Microfilament Proteins ,Molecular ,Actin remodeling ,Cofilin ,Actins ,Cell biology ,Other Physical Sciences ,Actin Cytoskeleton ,030104 developmental biology ,Treadmilling ,biology.protein ,Biochemistry and Cell Biology ,Generic health relevance ,MDia1 ,Biomolecular - Abstract
Cellular actin dynamics is an essential element of numerous cellular processes, such as cell motility, cell division and endocytosis. Actin’s involvement in these processes is mediated by many actin-binding proteins, among which the cofilin family plays unique and essential role in accelerating actin treadmilling in filamentous actin (F-actin) in a nucleotide-state dependent manner. Cofilin preferentially interacts with older filaments by recognizing time-dependent changes in F-actin structure associated with the hydrolysis of ATP and release of inorganic phosphate (Pi) from the nucleotide cleft of actin. The structure of cofilin on F-actin and the details of the intermolecular interface remain poorly understood at atomic resolution. Here we report atomic-level characterization by magic angle spinning (MAS) NMR of the muscle isoform of human cofilin 2 (CFL2) bound to F-actin. We demonstrate that resonance assignments for the majority of atoms are readily accomplished and we derive the intermolecular interface between CFL2 and F-actin. The MAS NMR approach reported here establishes the foundation for atomic-resolution characterization of a broad range of actin-associated proteins bound to F-actin.
- Published
- 2017
33. Thermodynamic properties of the effector domains of MARTX toxins suggest their unfolding for translocation across the host membrane
- Author
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Dmitri S. Kudryashov, David B. Heisler, Andrew Zywiec, and Elena Kudryashova
- Subjects
Effector ,Biology ,medicine.disease_cause ,Microbiology ,Cysteine protease ,Molten globule ,Protein structure ,Biochemistry ,Vibrio cholerae ,Cytoplasm ,medicine ,Biophysics ,Protein folding ,Thermolabile ,Molecular Biology - Abstract
MARTX (multifunctional autoprocessing repeats-in-toxin) family toxins are produced by Vibrio cholerae, Vibrio vulnificus, Aeromonas hydrophila and other Gram-negative bacteria. Effector domains of MARTX toxins cross the cytoplasmic membrane of a host cell through a putative pore formed by the toxin's glycine-rich repeats. The structure of the pore is unknown and the translocation mechanism of the effector domains is poorly understood. We examined the thermodynamic stability of the effector domains of V. cholerae and A. hydrophila MARTX toxins to elucidate the mechanism of their translocation. We found that all but one domain in each toxin are thermodynamically unstable and several acquire a molten globule state near human physiological temperatures. Fusion of the most stable cysteine protease domain to the adjacent effector domain reduces its thermodynamic stability ∼ 1.4-fold (from D G H 2 O 21.8 to 16.1 kJ mol(-1) ). Precipitation of several individual domains due to thermal denaturation is reduced upon their fusion into multi-domain constructs. We speculate that low thermostability of the MARTX effector domains correlates with that of many other membrane-penetrating toxins and implies their unfolding for cell entry. This study extends the list of thermolabile bacterial toxins, suggesting that this quality is essential and could be susceptible for selective targeting of pathogenic toxins.
- Published
- 2014
34. Investigations into the Structure and Intermolecular Interface of Human Cofilin-2 Assembled on Actin Filaments by Magic Angle Spinning NMR
- Author
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Tatyana Polenova, Jenna Yehl, Jodi Kraus, Emil Reisler, Dmitri S. Kudryashov, and Elena Kudryashova
- Subjects
Materials science ,Chemical physics ,Interface (Java) ,Intermolecular force ,Biophysics ,Magic angle spinning ,Cofilin ,Actin - Published
- 2019
35. Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins
- Author
-
Mamuka Kvaratskhelia, Wuyuan Lu, Adam A. Strömstedt, Elena Kudryashova, Dmitri S. Kudryashov, and Pratibha C. Koneru
- Subjects
0301 basic medicine ,Protein Folding ,alpha-Defensins ,beta-Defensins ,Protein Conformation ,Angiogenesis ,animal diseases ,Potyvirus ,chemical and pharmacologic phenomena ,Inflammation ,Plasma protein binding ,Biology ,Article ,Viral Proteins ,Pharmaceutical Sciences ,03 medical and health sciences ,Immune system ,Simian foamy virus ,medicine ,Chemical Precipitation ,Protein Unfolding ,Multidisciplinary ,Innate immune system ,030102 biochemistry & molecular biology ,Protein Stability ,Pathogen-associated molecular pattern ,fungi ,respiratory system ,biochemical phenomena, metabolism, and nutrition ,Farmaceutiska vetenskaper ,3. Good health ,Cell biology ,030104 developmental biology ,Beta defensin ,Proteolysis ,Immunology ,HIV-1 ,Unfolded protein response ,Thermodynamics ,bacteria ,medicine.symptom ,Peptides ,Hydrophobic and Hydrophilic Interactions ,Protein Binding - Abstract
Human defensins are innate immune defense peptides with a remarkably broad repertoire of anti-pathogen activities. In addition to modulating immune response, inflammation and angiogenesis, disintegrating bacterial membranes and inactivating bacterial toxins, defensins are known to intercept various viruses at different stages of their life cycles, while remaining relatively benign towards human cells and proteins. Recently we have found that human defensins inactivate proteinaceous bacterial toxins by taking advantage of their low thermodynamic stability and acting as natural “anti-chaperones”, i.e. destabilizing the native conformation of the toxins. In the present study we tested various proteins produced by several viruses (HIV-1, PFV and TEV) and found them to be susceptible to destabilizing effects of human α-defensins HNP-1 and HD-5 and the synthetic θ-defensin RC-101, but not β-defensins hBD-1 and hBD-2 or structurally related plant-derived peptides. Defensin-induced unfolding promoted exposure of hydrophobic groups otherwise confined to the core of the viral proteins. This resulted in precipitation, an enhanced susceptibility to proteolytic cleavage and a loss of viral protein activities. We propose, that defensins recognize and target a common and essential physico-chemical property shared by many bacterial toxins and viral proteins – the intrinsically low thermodynamic protein stability.
- Published
- 2016
36. Pathogenic Mechanisms of Actin Cross-Linking Toxins: Peeling Away the Layers
- Author
-
Dmitri S. Kudryashov, Elena Kudryashova, and David B. Heisler
- Subjects
0301 basic medicine ,biology ,Chemistry ,Effector ,030106 microbiology ,Cell ,macromolecular substances ,medicine.disease_cause ,biology.organism_classification ,Actin cytoskeleton ,Vibrio ,Cell biology ,03 medical and health sciences ,medicine.anatomical_structure ,Cytoplasm ,Vibrio cholerae ,Formins ,medicine ,biology.protein ,Actin - Abstract
Actin cross-linking toxins are produced by Gram-negative bacteria from Vibrio and Aeromonas genera. The toxins were named actin cross-linking domains (ACD), since the first and most of the subsequently discovered ACDs were found as effector domains in larger MARTX and VgrG toxins. Among recognized human pathogens, ACD is produced by Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Upon delivery to the cytoplasm of a host cell, ACD covalently cross-links actin monomers into non-polymerizable actin oligomers of various lengths. Provided sufficient doses of toxin are delivered, most or all actin can be promptly cross-linked into non-functional oligomers, leading to cell rounding, detachment from the substrate and, in many cases, cell death. Recently, a deeper layer of ACD toxicity with a less obvious but more potent mechanism was discovered. According to this finding, low doses of the ACD-produced actin oligomers can actively disrupt the actin cytoskeleton by potently inhibiting essential actin assembly proteins, formins. The first layer of toxicity is direct (as actin is the immediate and the only target), passive (since ACD-cross-linked actin oligomers are toxic only because they are non-functional), and less potent (as bulk quantities of one of the most abundant cytoplasmic proteins, actin, have to be modified). The second mechanism is indirect (as major targets, formins, are not affected by ACD directly), active (because actin oligomers act as “secondary” toxins), and highly potent [as it affects scarce and essential actin-binding proteins (ABPs)].
- Published
- 2016
37. Satellite cell senescence underlies myopathy in a mouse model of limb-girdle muscular dystrophy 2H
- Author
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Irina Kramerova, Melissa J. Spencer, and Elena Kudryashova
- Subjects
Male ,Senescence ,Sarcopenia ,Satellite Cells, Skeletal Muscle ,Ubiquitin-Protein Ligases ,Primary Cell Culture ,Apoptosis ,Cell Count ,Biology ,Mice ,Phosphatidylinositol 3-Kinases ,Atrophy ,medicine ,Animals ,Regeneration ,Myocyte ,Muscular dystrophy ,Muscle, Skeletal ,Myopathy ,Cells, Cultured ,Cellular Senescence ,Mice, Knockout ,Genetics ,Mice, Inbred BALB C ,Myogenesis ,Cell Cycle ,Sumoylation ,Cell Differentiation ,General Medicine ,medicine.disease ,Protein Inhibitors of Activated STAT ,Muscle atrophy ,Cell biology ,Disease Models, Animal ,Muscular Dystrophies, Limb-Girdle ,medicine.symptom ,Proto-Oncogene Proteins c-akt ,Signal Transduction ,Research Article ,Limb-girdle muscular dystrophy - Abstract
Mutations in the E3 ubiquitin ligase tripartite motif-containing 32 (TRIM32) are responsible for the disease limb-girdle muscular dystrophy 2H (LGMD2H). Previously, we generated Trim32 knockout mice (Trim32–/– mice) and showed that they display a myopathic phenotype accompanied by neurogenic features. Here, we used these mice to investigate the muscle-specific defects arising from the absence of TRIM32, which underlie the myopathic phenotype. Using 2 models of induced atrophy, we showed that TRIM32 is dispensable for muscle atrophy. Conversely, TRIM32 was necessary for muscle regrowth after atrophy. Furthermore, TRIM32-deficient primary myoblasts underwent premature senescence and impaired myogenesis due to accumulation of PIAS4, an E3 SUMO ligase and TRIM32 substrate that was previously shown to be associated with senescence. Premature senescence of myoblasts was also observed in vivo in an atrophy/regrowth model. Trim32–/– muscles had substantially fewer activated satellite cells, increased PIAS4 levels, and growth failure compared with wild-type muscles. Moreover, Trim32–/– muscles exhibited features of premature sarcopenia, such as selective type II fast fiber atrophy. These results imply that premature senescence of muscle satellite cells is an underlying pathogenic feature of LGMD2H and reveal what we believe to be a new mechanism of muscular dystrophy associated with reductions in available satellite cells and premature sarcopenia.
- Published
- 2012
38. Double-strand break repair and recombination-dependent replication of DNA in bacteriophage T4 in the absence of UvsX recombinase: Replicative resolution pathway
- Author
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Svetlana V. Sizova, Lidia Plugina, V P Shcherbakov, Tamara Shcherbakova, and Elena Kudryashova
- Subjects
DNA Replication ,DNA Repair ,DNA repair ,Mutant ,Biology ,Biochemistry ,Genetic recombination ,Viral Proteins ,Gene Order ,Recombinase ,Holliday junction ,Bacteriophage T4 ,DNA Breaks, Double-Stranded ,Molecular Biology ,Crosses, Genetic ,Recombination, Genetic ,Genetics ,Models, Genetic ,Point mutation ,DNA replication ,Membrane Proteins ,Cell Biology ,Double Strand Break Repair ,DNA-Binding Proteins ,Rec A Recombinases ,Mutation - Abstract
The effects of mutations in bacteriophage T4 genes uvsX and 49 on the double-strand break (DSB)-promoted recombination were studied in crosses, in which DSBs were induced site-specifically within the rIIB gene by SegC endonuclease in the DNA of only one of the parents. Frequency of rII+ recombinants was measured in two-factor crosses of the type i×ets1 and in three-factor crosses of the type i×ets1 a6, where ets1 is an insertion in the rIIB gene carrying the cleavage site for SegC; i's are rIIB or rIIA point mutations located at various distances (12-2040 bp) from the ets1 site, and a6 is rIIA point mutation located at 2040 bp from ets1. The frequency/distance relationships were obtained in crosses of the wild-type phage and of the amber mutant S17 (gene uvsX) and the double mutant S17 E727 (genes uvsX and 49). These data provide information about the frequency and distance distribution of the single-exchange (splices) and double-exchange (patches) events. The extended variant of the splice/patch coupling (SPC) model of recombination, which includes transition to the replication resolution (RR) alternative is substantiated and used for interpretation of the frequency/distance relationships. We conclude that the uvsX mutant executes recombination-dependent replication but does it by a qualitatively different way. In the absence of UvsX function, the DSB repair runs largely through the RR subpathway because of inability of the mutant to form a Holliday junction. In the two-factor crosses, the double uvsX 49- is recombinationally more proficient than the single uvsX mutant (partial suppression of the uvsX deficiency), while the patch-related double exchanges are virtually eliminated in this background.
- Published
- 2012
39. Impaired calcium calmodulin kinase signaling and muscle adaptation response in the absence of calpain 3
- Author
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Natalia Ermolova, Irina Kramerova, Melissa J. Spencer, A. López de Munain, Amets Sáenz, Elena Kudryashova, and Oihane Jaka
- Subjects
Adult ,Male ,medicine.medical_specialty ,Calmodulin ,Down-Regulation ,Muscle Proteins ,Mice ,Young Adult ,Ca2+/calmodulin-dependent protein kinase ,Internal medicine ,Myosin ,Genetics ,medicine ,Animals ,Humans ,Muscle, Skeletal ,Molecular Biology ,CAMK ,Genetics (clinical) ,Mice, Knockout ,biology ,Calpain ,Ryanodine receptor ,Muscle adaptation ,Skeletal muscle ,Articles ,General Medicine ,Middle Aged ,Endocrinology ,medicine.anatomical_structure ,Muscular Dystrophies, Limb-Girdle ,biology.protein ,Calcium ,Female ,Calcium-Calmodulin-Dependent Protein Kinase Type 2 ,Signal Transduction - Abstract
Mutations in the non-lysosomal, cysteine protease calpain 3 (CAPN3) result in the disease limb girdle muscular dystrophy type 2A (LGMD2A). CAPN3 is localized to several subcellular compartments, including triads, where it plays a structural, rather than a proteolytic, role. In the absence of CAPN3, several triad components are reduced, including the major Ca(2+) release channel, ryanodine receptor (RyR). Furthermore, Ca(2+) release upon excitation is impaired in the absence of CAPN3. In the present study, we show that Ca-calmodulin protein kinase II (CaMKII) signaling is compromised in CAPN3 knockout (C3KO) mice. The CaMK pathway has been previously implicated in promoting the slow skeletal muscle phenotype. As expected, the decrease in CaMKII signaling that was observed in the absence of CAPN3 is associated with a reduction in the slow versus fast muscle fiber phenotype. We show that muscles of WT mice subjected to exercise training activate the CaMKII signaling pathway and increase expression of the slow form of myosin; however, muscles of C3KO mice do not exhibit these adaptive changes to exercise. These data strongly suggest that skeletal muscle's adaptive response to functional demand is compromised in the absence of CAPN3. In agreement with our mouse studies, RyR levels were also decreased in biopsies from LGMD2A patients. Moreover, we observed a preferential pathological involvement of slow fibers in LGMD2A biopsies. Thus, impaired CaMKII signaling and, as a result, a weakened muscle adaptation response identify a novel mechanism that may underlie LGMD2A and suggest a pharmacological target that should be explored for therapy.
- Published
- 2012
40. Pathogenity of some limb girdle muscular dystrophy mutations can result from reduced anchorage to myofibrils and altered stability of calpain 3
- Author
-
Julio L. Vergara, Marino DiFranco, Irina Kramerova, Natalia Ermolova, Melissa J. Spencer, and Elena Kudryashova
- Subjects
Male ,Blotting, Western ,Mutation, Missense ,Mice, Transgenic ,Mice ,Myofibrils ,Genetics ,medicine ,Animals ,Muscular dystrophy ,Molecular Biology ,Genetics (clinical) ,biology ,Calpain ,Reverse Transcriptase Polymerase Chain Reaction ,Proteolytic enzymes ,Skeletal muscle ,Articles ,General Medicine ,medicine.disease ,Immunohistochemistry ,Cell biology ,medicine.anatomical_structure ,Muscular Dystrophies, Limb-Girdle ,Biochemistry ,Mutation ,biology.protein ,Titin ,Titin binding ,Myofibril ,Limb-girdle muscular dystrophy - Abstract
Calpain 3 (CAPN3) is a muscle-specific, calcium-dependent proteinase that is mutated in Limb Girdle Muscle Dystrophy type 2A. Most pathogenic missense mutations in LGMD2A affect CAPN3's proteolytic activity; however, two mutations, D705G and R448H, retain activity but nevertheless cause muscular dystrophy. Previously, we showed that D705G and R448H mutations reduce CAPN3s ability to bind to titin in vitro. In this investigation, we tested the consequence of loss of titin binding in vivo and examined whether this loss can be an underlying pathogenic mechanism in LGMD2A. To address this question, we created transgenic mice that express R448H or D705G in muscles, on wild-type (WT) CAPN3 or knock-out background. Both mutants were readily expressed in insect cells, but when D705G was expressed in skeletal muscle, it was not stable enough to study. Moreover, the D705G mutation had a dominant negative effect on endogenous CAPN3 when expressed on a WT background. The R448H protein was stably expressed in muscles; however, it was more rapidly degraded in muscle extracts compared with WT CAPN3. Increased degradation of R448H was due to non-cysteine, cellular proteases acting on the autolytic sites of CAPN3, rather than autolysis. Fractionation experiments revealed a significant decrease of R448H from the myofibrillar fraction, likely due to the mutant's inability to bind titin. Our data suggest that R448H and D705G mutations affect both CAPN3s anchorage to titin and its stability. These studies reveal a novel mechanism by which mutations that spare enzymatic activity can still lead to calpainopathy.
- Published
- 2011
41. On the mutagenicity of homologous recombination and double-strand break repair in bacteriophage
- Author
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Elena Kudryashova, V P Shcherbakov, Plugina La, Svetlana V. Sizova, and Tamara Shcherbakova
- Subjects
DNA Replication ,DNA Repair ,DNA repair ,Molecular Sequence Data ,Biology ,Virus Replication ,medicine.disease_cause ,Biochemistry ,Genetic recombination ,Viral Proteins ,chemistry.chemical_compound ,Recombinase ,medicine ,Bacteriophage T4 ,DNA Breaks, Double-Stranded ,DNA Cleavage ,Molecular Biology ,Recombination, Genetic ,Genetics ,Mutation ,Endodeoxyribonucleases ,Base Sequence ,fungi ,DNA replication ,Membrane Proteins ,Cell Biology ,Double Strand Break Repair ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,chemistry ,DNA, Viral ,Homologous recombination ,DNA - Abstract
The double-strand break (DSB) repair via homologous recombination is generally construed as a high-fidelity process. However, some molecular genetic observations show that the recombination and the recombinational DSB repair may be mutagenic and even highly mutagenic. Here we developed an effective and precise method for studying the fidelity of DSB repair in vivo by combining DSBs produced site-specifically by the SegC endonuclease with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. The method is based on the comparison of the rate of reversion of rII mutation in the presence and in the absence of a DSB repair event initiated in the proximity of the mutation. We observed that DSB repair may moderately (up to 6-fold) increase the apparent reversion frequency, the effect of being dependent on the mutation structure. We also studied the effect of the T4 recombinase deficiency (amber mutation in the uvsX gene) on the fidelity of DSB repair. We observed that DSBs are still repaired via homologous recombination in the uvsX mutants, and the apparent fidelity of this repair is higher than that seen in the wild-type background. The mutator effect of the DSB repair may look unexpected given that most of the normal DNA synthesis in bacteriophage T4 is performed via a recombination-dependent replication (RDR) pathway, which is thought to be indistinguishable from DSB repair. There are three possible explanations for the observed mutagenicity of DSB repair: (1) the origin-dependent (early) DNA replication may be more accurate than the RDR; (2) the step of replication initiation may be more mutagenic than the process of elongation; and (3) the apparent mutagenicity may just reflect some non-randomness in the pool of replicating DNA, i.e., preferential replication of the sequences already involved in replication. We discuss the DSB repair pathway in the absence of UvsX recombinase.
- Published
- 2011
42. Deficiency of the E3 ubiquitin ligase TRIM32 in mice leads to a myopathy with a neurogenic component
- Author
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Jun Wu, Melissa J. Spencer, Leif A. Havton, and Elena Kudryashova
- Subjects
Neurogenesis ,Ubiquitin-Protein Ligases ,Biology ,medicine.disease_cause ,Mice ,Muscular Diseases ,Neurofilament Proteins ,Myosin ,Genetics ,medicine ,Animals ,Protein Isoforms ,Muscular dystrophy ,Muscle, Skeletal ,Myopathy ,Molecular Biology ,Myelin Sheath ,Genetics (clinical) ,Actin ,Mice, Knockout ,Mutation ,Myosin Heavy Chains ,Brain ,Skeletal muscle ,Articles ,General Medicine ,medicine.disease ,Molecular biology ,Axons ,Ubiquitin ligase ,Mice, Inbred C57BL ,Motor unit ,medicine.anatomical_structure ,biology.protein ,medicine.symptom ,Gene Deletion - Abstract
Limb-girdle muscular dystrophy type 2H (LGMD2H) and sarcotubular myopathy are hereditary skeletal muscle disorders caused by mutations in TRIM32. We previously identified TRIM32 as an E3 ubiquitin ligase that binds to myosin and ubiquitinates actin. To date four TRIM32 mutations have been linked to LGMD2H, all of which occur in the C-terminal NHL domains. Unexpectedly, a fifth mutation in the B-box of TRIM32 causes a completely different, multisystemic disorder, Bardet-Biedl syndrome type 11. It is not understood how allelic mutations in TRIM32 can create such diverse phenotypic outcomes. To generate a tool for elucidating the complex in vivo functions of TRIM32, we created the first murine Trim32 knock-out model (T32KO). Histological analysis of T32KO skeletal muscles revealed mild myopathic changes. Electron microscopy showed areas with Z-line streaming and a dilated sarcotubular system with vacuoles -- the latter being a prominent feature of sarcotubular myopathy. Therefore, our model replicates phenotypes of LGMD2H and sarcotubular myopathy. The level of Trim32 expression in normal mouse brain exceeds that observed in skeletal muscle by more than 100 times, as we demonstrated by real-time PCR. Intriguingly, analysis of T32KO neural tissue revealed a decreased concentration of neurofilaments and a reduction in myelinated motoraxon diameters. The axonal changes suggest a shift toward a slower motor unit type. Not surprisingly, T32KO soleus muscle expressed an elevated type I slow myosin isotype with a concomitant reduction in the type II fast myosin. These data suggest that muscular dystrophy due to TRIM32 mutations involves both neurogenic and myogenic characteristics.
- Published
- 2009
43. ACTIN-DIRECTED TOXIN. ACD toxin-produced actin oligomers poison formin-controlled actin polymerization
- Author
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David B, Heisler, Elena, Kudryashova, Dmitry O, Grinevich, Cristian, Suarez, Jonathan D, Winkelman, Konstantin G, Birukov, Sainath R, Kotha, Narasimham L, Parinandi, Dimitrios, Vavylonis, David R, Kovar, and Dmitri S, Kudryashov
- Subjects
Fetal Proteins ,Antigens, Bacterial ,Bacterial Toxins ,Microfilament Proteins ,Formins ,Nuclear Proteins ,macromolecular substances ,Actins ,Article ,Cell Line ,Polymerization ,Protein Structure, Tertiary ,Rats ,Animals ,Intestinal Mucosa - Abstract
The actin crosslinking domain (ACD) is an actin-specific toxin produced by several pathogens, including life-threatening spp. of Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Actin crosslinking by ACD is thought to lead to slow cytoskeleton failure owing to a gradual sequestration of actin in the form of nonfunctional oligomers. Here we found that ACD converted cytoplasmic actin into highly toxic oligomers that potently “poisoned” the ability of major actin assembly proteins, formins, to sustain actin polymerization. Thus, ACD can target the most abundant cellular protein by employing actin oligomers as secondary toxins to efficiently subvert cellular functions of actin while functioning at very low doses.
- Published
- 2015
44. Retrocyclins neutralize bacterial toxins by potentiating their unfolding
- Author
-
Wuyuan Lu, Elena Kudryashova, Dmitri S. Kudryashov, and Stephanie Seveau
- Subjects
Microbial toxins ,alpha-Defensins ,beta-Defensins ,Toxin ,Recombinant Fusion Proteins ,Bacterial Toxins ,Proteolytic degradation ,Cell Biology ,Biology ,medicine.disease_cause ,Biochemistry ,Article ,Microbiology ,Cell Line ,medicine ,Humans ,Molecular Biology ,Protein Unfolding - Abstract
Defensins are a class of immune peptides with a broad range of activities against bacterial, fungal and viral pathogens. Besides exerting direct anti-microbial activity via dis-organization of bacterial membranes, defensins are also able to neutralize various unrelated bacterial toxins. Recently, we have demonstrated that in the case of human α- and β-defensins, this later ability is achieved through exploiting toxins' marginal thermodynamic stability, i.e. defensins act as molecular anti-chaperones unfolding toxin molecules and exposing their hydrophobic regions and thus promoting toxin precipitation and inactivation [Kudryashova et al. (2014) Immunity 41, 709-721]. Retrocyclins (RCs) are humanized synthetic θ-defensin peptides that possess unique cyclic structure, differentiating them from α- and β-defensins. Importantly, RCs are more potent against some bacterial and viral pathogens and more stable than their linear counterparts. However, the mechanism of bacterial toxin inactivation by RCs is not known. In the present study, we demonstrate that RCs facilitate unfolding of bacterial toxins. Using differential scanning fluorimetry (DSF), limited proteolysis and collisional quenching of internal tryptophan fluorescence, we show that hydrophobic regions of toxins normally buried in the molecule interior become more exposed to solvents and accessible to proteolytic cleavage in the presence of RCs. The RC-induced unfolding of toxins led to their precipitation and abrogated activity. Toxin inactivation by RCs was strongly diminished under reducing conditions, but preserved at physiological salt and serum concentrations. Therefore, despite significant structural diversity, α-, β- and θ-defensins employ similar mechanisms of toxin inactivation, which may be shared by anti-microbial peptides from other families.
- Published
- 2015
45. Double-Strand Break Repair in Bacteriophage T4: Recombination Effects of 3′–5′ Exonuclease Mutations
- Author
-
Plugina La, Tamara Shcherbakova, Svetlana T. Sizova, Elena Kudryashova, and V P Shcherbakov
- Subjects
Exonucleases ,Recombination, Genetic ,Genetics ,Exonuclease ,Deoxyribonucleases ,biology ,DNA polymerase ,DNA polymerase II ,Point mutation ,Mutant ,DNA ,DNA Polymerase II ,Investigations ,Molecular biology ,chemistry.chemical_compound ,chemistry ,Mutation ,Escherichia coli ,3'-5' Exonuclease ,biology.protein ,Bacteriophage T4 ,Proofreading ,DNA Breaks, Double-Stranded - Abstract
The role of 3′–5′ exonucleases in double-strand break (DSB)-promoted recombination was studied in crosses of bacteriophage T4, in which DSBs were induced site specifically within the rIIB gene by SegC endonuclease in the DNA of only one of the parents. Frequency of rII+ recombinants was measured in two-factor crosses of the type i × ets1, where ets1 designates an insertion in the rIIB gene carrying the cleavage site for SegC and i's are rIIB or rIIA point mutations located at various distances (12–2040 bp) from the ets1 site. The frequency/distance relationship was obtained in crosses of the wild-type phage and dexA1 (deficiency in deoxyribonuclease A), D219A (deficiency in the proofreading exonuclease of DNA polymerase), and tsL42 (antimutator allele of DNA polymerase) mutants. In all the mutants, recombinant frequency in crosses with the i-markers located at 12 and 33 bp from ets1 was significantly enhanced, implying better preservation of 3′-terminal sequences at the ends of the broken DNA. The effects of dexA1 and D219A were additive, suggesting an independent action of the corresponding nucleases in the DSB repair pathway. The recombination enhancement in the dexA1 mutant was limited to short distances (
- Published
- 2006
46. Calpain 3 participates in sarcomere remodeling by acting upstream of the ubiquitin–proteasome pathway
- Author
-
Elena Kudryashova, Gayathri Venkatraman, Irina Kramerova, and Melissa J. Spencer
- Subjects
Male ,Sarcomeres ,Proteasome Endopeptidase Complex ,Class I Phosphatidylinositol 3-Kinases ,Down-Regulation ,Muscle Proteins ,Protein degradation ,Sarcomere ,Mice ,Phosphatidylinositol 3-Kinases ,Ubiquitin ,Multienzyme Complexes ,Heat shock protein ,Genetics ,Animals ,Insulin-Like Growth Factor I ,Molecular Biology ,Heat-Shock Proteins ,Genetics (clinical) ,Mice, Knockout ,biology ,Calpain ,Protein turnover ,General Medicine ,Immunohistochemistry ,Cell biology ,Hindlimb Suspension ,Muscular Dystrophies, Limb-Girdle ,Proteasome ,Biochemistry ,biology.protein ,Myofibril - Abstract
Mutations in the non-lysosomal cysteine protease calpain 3 cause limb-girdle muscular dystrophy type 2A (LGMD2A). Our previous studies of the calpain 3 knockout mouse (C3KO) suggested a role for calpain 3 in sarcomere formation and remodeling. Calpain 3 may mediate remodeling by cleavage and release of myofibrillar proteins, targeting them for ubiquitination and proteasomal degradation. Loss of proper protein turnover may be the basis for this muscle disease. To test this hypothesis in vivo, we used an experimental model of hindlimb unloading and reloading that has been shown to induce sarcomere remodeling. We showed that the rate of atrophy and especially the rate of growth are decreased in C3KO muscles under conditions promoting sarcomere remodeling. In wild-type mice, an elevated level of ubiquitinated proteins was observed during muscle reloading, which is presumably necessary to remove atrophy-specific and damaged proteins. This increase in ubiquitination correlated with an increase in calpain 3 expression. C3KO muscles did not show any increase in ubiquitination at the reloading stage, suggesting that calpain 3 is necessary for ubiquitination and that it acts upstream of the ubiquitination machinery. We found upregulation of heat shock proteins in C3KO muscles following challenge with a physiological condition that requires highly increased protein degradation. Furthermore, old C3KO mice show evidence of insoluble protein aggregate formation in skeletal muscles. These studies suggest that accumulation of aged and damaged proteins can lead to cellular toxicity and a cell stress response in C3KO muscles, and that these characteristics are pathological features of LGMD2A.
- Published
- 2005
47. The E3 ubiquitin ligase TRIM32 regulates myoblast proliferation by controlling turnover of NDRG2
- Author
-
Michael J. Haykinson, Elena Kudryashova, Nuraly K. Avliyakulov, Irina Kramerova, Melissa J. Spencer, and Ekaterina Mokhonova
- Subjects
Myoblast proliferation ,Medical and Health Sciences ,Muscular Dystrophies ,Myoblasts ,Two-Dimensional Difference Gel Electrophoresis ,Limb-Girdle ,Gene Knockout Techniques ,Mice ,Ubiquitin ,Myocyte ,Genetics (clinical) ,Genetics & Heredity ,chemistry.chemical_classification ,Mice, Knockout ,biology ,Myogenesis ,Cell Cycle ,Adaptor Proteins ,General Medicine ,Articles ,Biological Sciences ,Ubiquitin ligase ,Up-Regulation ,medicine.anatomical_structure ,Stem Cell Research - Nonembryonic - Non-Human ,Knockout ,Intellectual and Developmental Disabilities (IDD) ,1.1 Normal biological development and functioning ,Ubiquitin-Protein Ligases ,Rare Diseases ,Underpinning research ,Genetics ,medicine ,Animals ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,Cell Proliferation ,DNA ligase ,Animal ,Cell growth ,Signal Transducing ,Skeletal muscle ,Proteins ,Stem Cell Research ,Molecular biology ,Brain Disorders ,Disease Models, Animal ,chemistry ,Muscular Dystrophies, Limb-Girdle ,Musculoskeletal ,Disease Models ,biology.protein - Abstract
Limb girdle muscular dystrophy 2H is caused by mutations in the gene encoding the E3 ubiquitin ligase, TRIM32. Previously, we generated and characterized a Trim32 knockout mouse (T32KO) that displays both neurogenic and myopathic features. The myopathy in these mice is attributable to impaired muscle growth, associated with satellite cell senescence and premature sarcopenia. This satellite cell senescence is due to accumulation of the SUMO ligase PIASy, a substrate of TRIM32. The goal of this investigation was to identify additional substrates of TRIM32 using 2D fluorescence difference gel electrophoresis (2D-DIGE) in order to further explore its role in skeletal muscle. Because TRIM32 is an E3 ubiquitin ligase, we reasoned that TRIM32's substrates would accumulate in its absence. 2D-DIGE identified 19 proteins that accumulate in muscles from the T32KO mouse. We focused on two of these proteins, NDRG2 and TRIM72, due to their putative roles in myoblast proliferation and myogenesis. Follow-up analysis confirmed that both proteins were ubiquitinated by TRIM32 in vitro; however, only NDRG2 accumulated in skeletal muscle and myoblasts in the absence of TRIM32. NDRG2 overexpression in myoblasts led to reduced cell proliferation and delayed cell cycle withdrawal during differentiation. Thus, we identified NDRG2 as a novel target for TRIM32; these findings further corroborate the hypothesis that TRIM32 is involved in control of myogenic cells proliferation and differentiation.
- Published
- 2014
48. Split and Splice' Approach for Highly Selective Targeting of Human NSCLC Tumors'
- Author
-
Dmitri S. Kudryashov and Elena Kudryashova
- Subjects
Microbial toxins ,Biochemistry ,In vivo ,Solubilization ,Lysine ,splice ,Biology ,Highly selective ,Intein ,Cysteine - Abstract
The goal of the current proposal was to develop a novel split and splice approach for highly selective targeting and elimination of NSCLC cells. We have optimized DnaENpu intein for Stx2A-specific intracellular delivery mechanism by replacing four lysine residues in its C-terminal part with arginines and confirming that the resulted mutated intein retains its trans-splicing activity in vivo. We have confirmed that fusion of the C-terminal DnaENpu split intein does not interfere with cellular delivery of bacterial toxins; whereas the N-terminal DnaENpu intein slows down the delivery. The work is in progress to optimize properties of the N-terminal DnaENpu intein to allow for more efficient translocation of the desired toxins by removing two cysteine residues and affecting thermodynamic properties of this part of the split intein. The most significant hindrance that we have observed is low solubility of the C-terminal Stx2A split intein constructs and their inability to form a complex with Stx2B subunits. Currently we are employing several parallel strategies to optimize solubility of the above protein constructs via co-expression and refolding/solubilization strategies.
- Published
- 2014
49. Thermodynamic properties of the effector domains of MARTX toxins suggest their unfolding for translocation across the host membrane
- Author
-
Elena, Kudryashova, David, Heisler, Andrew, Zywiec, and Dmitri S, Kudryashov
- Subjects
Protein Folding ,Protein Transport ,Bacterial Toxins ,Humans ,Thermodynamics ,Vibrio cholerae ,Aeromonas hydrophila ,Protein Structure, Tertiary - Abstract
MARTX (multifunctional autoprocessing repeats-in-toxin) family toxins are produced by Vibrio cholerae, Vibrio vulnificus, Aeromonas hydrophila and other Gram-negative bacteria. Effector domains of MARTX toxins cross the cytoplasmic membrane of a host cell through a putative pore formed by the toxin's glycine-rich repeats. The structure of the pore is unknown and the translocation mechanism of the effector domains is poorly understood. We examined the thermodynamic stability of the effector domains of V. cholerae and A. hydrophila MARTX toxins to elucidate the mechanism of their translocation. We found that all but one domain in each toxin are thermodynamically unstable and several acquire a molten globule state near human physiological temperatures. Fusion of the most stable cysteine protease domain to the adjacent effector domain reduces its thermodynamic stability ∼ 1.4-fold (from D G H 2 O 21.8 to 16.1 kJ mol(-1) ). Precipitation of several individual domains due to thermal denaturation is reduced upon their fusion into multi-domain constructs. We speculate that low thermostability of the MARTX effector domains correlates with that of many other membrane-penetrating toxins and implies their unfolding for cell entry. This study extends the list of thermolabile bacterial toxins, suggesting that this quality is essential and could be susceptible for selective targeting of pathogenic toxins.
- Published
- 2014
50. Double-strand break repair and genetic recombination in topoisomerase and primase mutants of bacteriophage T4
- Author
-
Elena Kudryashova and V P Shcherbakov
- Subjects
Genetics ,Recombination, Genetic ,Topoisomerase ,Mutant ,Recombinational DNA Repair ,Cell Biology ,DNA Primase ,Biology ,Biochemistry ,Molecular biology ,Genetic recombination ,law.invention ,Endonuclease ,Viral Proteins ,law ,Mutation ,Recombinant DNA ,biology.protein ,Bacteriophage T4 ,Primase ,Molecular Biology ,Gene ,Recombination ,DNA Topoisomerases - Abstract
Recombinational analysis Genetic recombination Gene 39 Gene 61 Topoisomerase II Primase a b s t r a c t The effects of primase and topoisomerase II deficiency on the double-strand break (DSB) repair and genetic recombination in bacteriophage T4 were studied in vivo using focused recombination. Site-specific DSBs were induced by SegC endonuclease in the rIIB gene of one of the parents. The frequency/distance relationship was determined in crosses of the wild-type phage, topoisomerase II mutant amN116 (gene 39), and primase mutant E219 (gene 61). Ordinary two-factor (i × j) and three-factor (i k × j) crosses between point rII mutations were also performed. These data provide information about the frequency and distance distribution of the single-exchange (splice) and double-exchange (patch) events. In two-factor crosses ets1 × i, the topoisomerase and primase mutants had similar recombinant frequencies in crosses at ets1–i distances longer than 1000 bp, comprising about 80% of the corresponding wild-type values. They, however, differ remarkably in crosses at shorter distances. In the primase mutant, the recombinant frequencies are similar to those in the wild-type crosses at distances less than 100 bp, being a bit diminished at longer distances. In two-factor crosses ets1 × i of the topoisomerase mutant, the recombinant frequencies were reduced ten-fold at the shortest distances. In three-factor crosses a6 ets1 × i, where we measure patch-related recombination, the primase mutant was quite proficient across the entire range of distances. The topoisomerase mutant crosses demonstrated virtually complete absence of rII+ recombinants at distances up to 33 bp, with the frequencies increasing steadily at longer distances. The data were interpreted as follows. The primase mutant is fully recombinationproficient. An obvious difference from the wild-type state is some shortage of EndoVII function leading to prolonged existence of HJs and thus stretched out ds-branch migration. This is also true for the topoisomerase mutant. However, the latter is deficient in the ss-branch migration step of the DSB repair pathway and partially deficient in HJ initiation. In apparent contradiction to their effects on the DSB-induced sitespecific recombination, the topoisomerase and primase mutants demonstrated about 3–8-fold increase in the recombinant frequencies in the ordinary crosses, with the recombination running exclusively via patches. This implies that most of the spontaneous recombination events are not initiated by dsDNA ends in these mutants.
- Published
- 2014
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