Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins During Ex Vivo Osteoblast Differentiation of Human Stromal Stem Cells*

Science.gov (United States)

Kristensen, Lars P.; Chen, Li; Nielsen, Maria Overbeck; Qanie, Diyako W.; Kratchmarova, Irina; Kassem, Moustapha; Andersen, Jens S.

2012-01-01

It is well established that bone forming cells (osteoblasts) secrete proteins with autocrine, paracrine, and endocrine function. However, the identity and functional role for the majority of these secreted and differentially expressed proteins during the osteoblast (OB) differentiation process, is not fully established. To address these questions, we quantified the temporal dynamics of the human stromal (mesenchymal, skeletal) stem cell (hMSC) secretome during ex vivo OB differentiation using stable isotope labeling by amino acids in cell culture (SILAC). In addition, we employed pulsed SILAC labeling to distinguish genuine secreted proteins from intracellular contaminants. We identified 466 potentially secreted proteins that were quantified at 5 time-points during 14-days ex vivo OB differentiation including 41 proteins known to be involved in OB functions. Among these, 315 proteins exhibited more than 2-fold up or down-regulation. The pulsed SILAC method revealed a strong correlation between the fraction of isotope labeling and the subset of proteins known to be secreted and involved in OB differentiation. We verified SILAC data using qRT-PCR analysis of 9 identified potential novel regulators of OB differentiation. Furthermore, we studied the biological effects of one of these proteins, the hormone stanniocalcin 2 (STC2) and demonstrated its autocrine effects in enhancing osteoblastic differentiation of hMSC. In conclusion, combining complete and pulsed SILAC labeling facilitated the identification of novel factors produced by hMSC with potential role in OB differentiation. Our study demonstrates that the secretome of osteoblastic cells is more complex than previously reported and supports the emerging evidence that osteoblastic cells secrete proteins with endocrine functions and regulate cellular processes beyond bone formation. PMID:22801418

Conformational dynamics data bank: a database for conformational dynamics of proteins and supramolecular protein assemblies.

Science.gov (United States)

Kim, Do-Nyun; Altschuler, Josiah; Strong, Campbell; McGill, Gaël; Bathe, Mark

2011-01-01

The conformational dynamics data bank (CDDB, http://www.cdyn.org) is a database that aims to provide comprehensive results on the conformational dynamics of high molecular weight proteins and protein assemblies. Analysis is performed using a recently introduced coarse-grained computational approach that is applied to the majority of structures present in the electron microscopy data bank (EMDB). Results include equilibrium thermal fluctuations and elastic strain energy distributions that identify rigid versus flexible protein domains generally, as well as those associated with specific functional transitions, and correlations in molecular motions that identify molecular regions that are highly coupled dynamically, with implications for allosteric mechanisms. A practical web-based search interface enables users to easily collect conformational dynamics data in various formats. The data bank is maintained and updated automatically to include conformational dynamics results for new structural entries as they become available in the EMDB. The CDDB complements static structural information to facilitate the investigation and interpretation of the biological function of proteins and protein assemblies essential to cell function.

Long-Term Dynamics of Autonomous Fractional Differential Equations

Science.gov (United States)

Liu, Tao; Xu, Wei; Xu, Yong; Han, Qun

This paper aims to investigate long-term dynamic behaviors of autonomous fractional differential equations with effective numerical method. The long-term dynamic behaviors predict where systems are heading after long-term evolution. We make some modification and transplant cell mapping methods to autonomous fractional differential equations. The mapping time duration of cell mapping is enlarged to deal with the long memory effect. Three illustrative examples, i.e. fractional Lotka-Volterra equation, fractional van der Pol oscillator and fractional Duffing equation, are studied with our revised generalized cell mapping method. We obtain long-term dynamics, such as attractors, basins of attraction, and saddles. Compared with some existing stability and numerical results, the validity of our method is verified. Furthermore, we find that the fractional order has its effect on the long-term dynamics of autonomous fractional differential equations.

Differential equations a dynamical systems approach ordinary differential equations

CERN Document Server

Hubbard, John H

1991-01-01

This is a corrected third printing of the first part of the text Differential Equations: A Dynamical Systems Approach written by John Hubbard and Beverly West. The authors' main emphasis in this book is on ordinary differential equations. The book is most appropriate for upper level undergraduate and graduate students in the fields of mathematics, engineering, and applied mathematics, as well as the life sciences, physics and economics. Traditional courses on differential equations focus on techniques leading to solutions. Yet most differential equations do not admit solutions which can be written in elementary terms. The authors have taken the view that a differential equations defines functions; the object of the theory is to understand the behavior of these functions. The tools the authors use include qualitative and numerical methods besides the traditional analytic methods. The companion software, MacMath, is designed to bring these notions to life.

Introduction to differential equations with dynamical systems

CERN Document Server

Campbell, Stephen L

2011-01-01

Many textbooks on differential equations are written to be interesting to the teacher rather than the student. Introduction to Differential Equations with Dynamical Systems is directed toward students. This concise and up-to-date textbook addresses the challenges that undergraduate mathematics, engineering, and science students experience during a first course on differential equations. And, while covering all the standard parts of the subject, the book emphasizes linear constant coefficient equations and applications, including the topics essential to engineering students. Stephen Campbell and Richard Haberman--using carefully worded derivations, elementary explanations, and examples, exercises, and figures rather than theorems and proofs--have written a book that makes learning and teaching differential equations easier and more relevant. The book also presents elementary dynamical systems in a unique and flexible way that is suitable for all courses, regardless of length.

Dynamics based alignment of proteins: an alternative approach to quantify dynamic similarity

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Lyngsø Rune

2010-04-01

Full Text Available Abstract Background The dynamic motions of many proteins are central to their function. It therefore follows that the dynamic requirements of a protein are evolutionary constrained. In order to assess and quantify this, one needs to compare the dynamic motions of different proteins. Comparing the dynamics of distinct proteins may also provide insight into how protein motions are modified by variations in sequence and, consequently, by structure. The optimal way of comparing complex molecular motions is, however, far from trivial. The majority of comparative molecular dynamics studies performed to date relied upon prior sequence or structural alignment to define which residues were equivalent in 3-dimensional space. Results Here we discuss an alternative methodology for comparative molecular dynamics that does not require any prior alignment information. We show it is possible to align proteins based solely on their dynamics and that we can use these dynamics-based alignments to quantify the dynamic similarity of proteins. Our method was tested on 10 representative members of the PDZ domain family. Conclusions As a result of creating pair-wise dynamics-based alignments of PDZ domains, we have found evolutionarily conserved patterns in their backbone dynamics. The dynamic similarity of PDZ domains is highly correlated with their structural similarity as calculated with Dali. However, significant differences in their dynamics can be detected indicating that sequence has a more refined role to play in protein dynamics than just dictating the overall fold. We suggest that the method should be generally applicable.

Effects of N-glycosylation on protein conformation and dynamics: Protein Data Bank analysis and molecular dynamics simulation study.

Science.gov (United States)

Lee, Hui Sun; Qi, Yifei; Im, Wonpil

2015-03-09

N-linked glycosylation is one of the most important, chemically complex, and ubiquitous post-translational modifications in all eukaryotes. The N-glycans that are covalently linked to proteins are involved in numerous biological processes. There is considerable interest in developments of general approaches to predict the structural consequences of site-specific glycosylation and to understand how these effects can be exploited in protein design with advantageous properties. In this study, the impacts of N-glycans on protein structure and dynamics are systematically investigated using an integrated computational approach of the Protein Data Bank structure analysis and atomistic molecular dynamics simulations of glycosylated and deglycosylated proteins. Our study reveals that N-glycosylation does not induce significant changes in protein structure, but decreases protein dynamics, likely leading to an increase in protein stability. Overall, these results suggest not only a common role of glycosylation in proteins, but also a need for certain proteins to be properly glycosylated to gain their intrinsic dynamic properties.

Dynamic data analysis modeling data with differential equations

CERN Document Server

Ramsay, James

2017-01-01

This text focuses on the use of smoothing methods for developing and estimating differential equations following recent developments in functional data analysis and building on techniques described in Ramsay and Silverman (2005) Functional Data Analysis. The central concept of a dynamical system as a buffer that translates sudden changes in input into smooth controlled output responses has led to applications of previously analyzed data, opening up entirely new opportunities for dynamical systems. The technical level has been kept low so that those with little or no exposure to differential equations as modeling objects can be brought into this data analysis landscape. There are already many texts on the mathematical properties of ordinary differential equations, or dynamic models, and there is a large literature distributed over many fields on models for real world processes consisting of differential equations. However, a researcher interested in fitting such a model to data, or a statistician interested in...

Hybrid Differential Dynamic Programming with Stochastic Search

Science.gov (United States)

Aziz, Jonathan; Parker, Jeffrey; Englander, Jacob

2016-01-01

Differential dynamic programming (DDP) has been demonstrated as a viable approach to low-thrust trajectory optimization, namely with the recent success of NASAs Dawn mission. The Dawn trajectory was designed with the DDP-based Static Dynamic Optimal Control algorithm used in the Mystic software. Another recently developed method, Hybrid Differential Dynamic Programming (HDDP) is a variant of the standard DDP formulation that leverages both first-order and second-order state transition matrices in addition to nonlinear programming (NLP) techniques. Areas of improvement over standard DDP include constraint handling, convergence properties, continuous dynamics, and multi-phase capability. DDP is a gradient based method and will converge to a solution nearby an initial guess. In this study, monotonic basin hopping (MBH) is employed as a stochastic search method to overcome this limitation, by augmenting the HDDP algorithm for a wider search of the solution space.

Differential scanning microcalorimetry of intrinsically disordered proteins.

Science.gov (United States)

Permyakov, Sergei E

2012-01-01

Ultrasensitive differential scanning calorimetry (DSC) is an indispensable thermophysical technique enabling to get direct information on enthalpies accompanying heating/cooling of dilute biopolymer solutions. The thermal dependence of protein heat capacity extracted from DSC data is a valuable source of information on intrinsic disorder level of a protein. Application details and limitations of DSC technique in exploration of protein intrinsic disorder are described.

Non-interacting surface solvation and dynamics in protein-protein interactions

NARCIS (Netherlands)

Visscher, Koen M.; Kastritis, Panagiotis L.|info:eu-repo/dai/nl/315886668; Bonvin, Alexandre M J J|info:eu-repo/dai/nl/113691238

2015-01-01

Protein-protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure-based drug design methods, as well as design of de novo

Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins during ex vivo Osteoblast Differentiation of Human Stromal Stem Cells

DEFF Research Database (Denmark)

Kristensen, Lars P; Chen, Li; Nielsen, Maria Overbeck

2012-01-01

, is not fully established. To address these questions, we quantified the temporal dynamics of the human stromal (mesenchymal, skeletal) stem cell (hMSC) secretome during ex vivo OB differentiation using stable isotope labeling by amino acids in cell culture (SILAC). In addition, we employed pulsed SILAC...... the identification of novel factors produced by hMSC with potential role in OB differentiation. Our study demonstrates that the secretome of osteoblastic cells is more complex than previously reported and supports the emerging evidence that osteoblastic cells secrete proteins with endocrine functions and regulate...... regulators of OB differentiation. Furthermore, we studied the biological effects of one of these proteins, the hormone stanniocalcin 2 (STC2) and demonstrated its autocrine effects in enhancing osteoblastic differentiation of hMSC. In conclusion, combining complete and pulsed SILAC labeling facilitated...

Proteinuria: The diagnostic strategy based on urine proteins differentiation

Directory of Open Access Journals (Sweden)

Stojimirović Biljana B.

2004-01-01

Full Text Available Basal glomerular membrane represents mechanical and electrical barrier for passing of the plasma proteins. Mechanical barrier is composed of cylindrical pores and filtration fissure, and negative layer charge in exterior and interior side of basal glomerular membrane, made of heparan sulphate and sialoglicoproteine, provides certain electrical barrier. Diagnostic strategy based on different serum and urine proteins enables the differentiation of various types of proteinuria. Depending on etiology of proteinuria it can be prerenal, renal and postrenal. By analyzing albumin, armicroglobulin, immunoglobulin G and armacroglobulin, together with total protein in urine, it is possible to detect and differentiate causes of prerenal, renal (glomerular, tubular, glomerulo-tubular and postrenal proteinuria. The adequate and early differentiation of proteinuria type is of an immense diagnostic and therapeutic importance.

The lie-algebraic structures and integrability of differential and differential-difference nonlinear dynamical systems

International Nuclear Information System (INIS)

Prykarpatsky, A.K.; Blackmore, D.L.; Bogolubov, N.N. Jr.

2007-05-01

The infinite-dimensional operator Lie algebras of the related integrable nonlocal differential-difference dynamical systems are treated as their hidden symmetries. As a result of their dimerization the Lax type representations for both local differential-difference equations and nonlocal ones are obtained. An alternative approach to the Lie-algebraic interpretation of the integrable local differential-difference systems is also proposed. The Hamiltonian representation for a hierarchy of Lax type equations on a dual space to the centrally extended Lie algebra of integro-differential operators with matrix-valued coefficients coupled with suitable eigenfunctions and adjoint eigenfunctions evolutions of associated spectral problems is obtained by means of a specially constructed Baecklund transformation. The Hamiltonian description for the corresponding set of additional symmetry hierarchies is represented. The relation of these hierarchies with Lax type integrable (3+1)-dimensional nonlinear dynamical systems and their triple Lax type linearizations is analyzed. The Lie-algebraic structures, related with centrally extended current operator Lie algebras are discussed with respect to constructing new nonlinear integrable dynamical systems on functional manifolds and super-manifolds. Special Poisson structures and related with them factorized integrable operator dynamical systems having interesting applications in modern mathematical physics, quantum computing mathematics and other fields are constructed. The previous purely computational results are explained within the approach developed. (author)

Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation

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Srsen Vlastimil

2009-04-01

Full Text Available Abstract Background Muscle fibres are formed by elongation and fusion of myoblasts into myotubes. During this differentiation process, the cytoskeleton is reorganized, and proteins of the centrosome re-localize to the surface of the nucleus. The exact timing of this event, and the underlying molecular mechanisms are still poorly understood. Results We performed studies on mouse myoblast cell lines that were induced to differentiate in culture, to characterize the early events of centrosome protein re-localization. We demonstrate that this re-localization occurs already at the single cell stage, prior to fusion into myotubes. Centrosome proteins that accumulate at the nuclear surface form an insoluble matrix that can be reversibly disassembled if isolated nuclei are exposed to mitotic cytoplasm from Xenopus egg extract. Our microscopy data suggest that this perinuclear matrix of centrosome proteins consists of a system of interconnected fibrils. Conclusion Our data provide new insights into the reorganization of centrosome proteins during muscular differentiation, at the structural and biochemical level. Because we observe that centrosome protein re-localization occurs early during differentiation, we believe that it is of functional importance for the reorganization of the cytoskeleton in the differentiation process.

Protein dynamics by neutron scattering: The protein dynamical transition and the fragile-to-strong dynamical crossover in hydrated lysozyme

International Nuclear Information System (INIS)

Magazù, Salvatore; Migliardo, Federica; Benedetto, Antonio; Vertessy, Beata

2013-01-01

Highlights: • The role played by the instrumental energy resolution in neutron scattering is presented. • The effect of natural bioprotectants on protein dynamics is shown. • A connection between the protein dynamical transition and the fragile-to-strong dynamical crossover is formulated. - Abstract: In this work Elastic Incoherent Neutron Scattering (EINS) results on lysozyme water mixtures in absence and in presence of bioprotectant systems are presented. The EINS data have been collected by using the IN13 and the IN10 spectrometers at the Institut Laue-Langevin (ILL, Grenoble, France) allowing to evaluate the temperature behaviour of the mean square displacement and of the relaxation time for the investigated systems. The obtained experimental findings together with theoretical calculations allow to put into evidence the role played by the spectrometer resolution and to clarify the connexion between the registered protein dynamical transition, the system relaxation time, and the instrumental energy resolution

Navier-Stokes dynamics on a differential one-form

Science.gov (United States)

Story, Troy L.

2006-11-01

After transforming the Navier-Stokes dynamic equation into a characteristic differential one-form on an odd-dimensional differentiable manifold, exterior calculus is used to construct a pair of differential equations and tangent vector(vortex vector) characteristic of Hamiltonian geometry. A solution to the Navier-Stokes dynamic equation is then obtained by solving this pair of equations for the position x^k and the conjugate to the position bk as functions of time. The solution bk is shown to be divergence-free by contracting the differential 3-form corresponding to the divergence of the gradient of the velocity with a triple of tangent vectors, implying constraints on two of the tangent vectors for the system. Analysis of the solution bk shows it is bounded since it remains finite as | x^k | ->,, and is physically reasonable since the square of the gradient of the principal function is bounded. By contracting the characteristic differential one-form with the vortex vector, the Lagrangian is obtained.

Protein kinesis: The dynamics of protein trafficking and stability

Energy Technology Data Exchange (ETDEWEB)

NONE

1995-12-31

The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

Differential Protein Expressions in Virus-Infected and Uninfected Trichomonas vaginalis.

Science.gov (United States)

He, Ding; Pengtao, Gong; Ju, Yang; Jianhua, Li; He, Li; Guocai, Zhang; Xichen, Zhang

2017-04-01

Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis , we detected 2 strains of T. vaginalis ; the virus-infected (V + ) and uninfected (V - ) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V + compared with V - isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V + isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V + and V - isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

Water Dynamics in Protein Hydration Shells: The Molecular Origins of the Dynamical Perturbation

Science.gov (United States)

2014-01-01

Protein hydration shell dynamics play an important role in biochemical processes including protein folding, enzyme function, and molecular recognition. We present here a comparison of the reorientation dynamics of individual water molecules within the hydration shell of a series of globular proteins: acetylcholinesterase, subtilisin Carlsberg, lysozyme, and ubiquitin. Molecular dynamics simulations and analytical models are used to access site-resolved information on hydration shell dynamics and to elucidate the molecular origins of the dynamical perturbation of hydration shell water relative to bulk water. We show that all four proteins have very similar hydration shell dynamics, despite their wide range of sizes and functions, and differing secondary structures. We demonstrate that this arises from the similar local surface topology and surface chemical composition of the four proteins, and that such local factors alone are sufficient to rationalize the hydration shell dynamics. We propose that these conclusions can be generalized to a wide range of globular proteins. We also show that protein conformational fluctuations induce a dynamical heterogeneity within the hydration layer. We finally address the effect of confinement on hydration shell dynamics via a site-resolved analysis and connect our results to experiments via the calculation of two-dimensional infrared spectra. PMID:24479585

Identification of Differentially Abundant Proteins of Edwardsiella ictaluri during Iron Restriction.

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Pradeep R Dumpala

Full Text Available Edwardsiella ictaluri is a Gram-negative facultative anaerobe intracellular bacterium that causes enteric septicemia in channel catfish. Iron is an essential inorganic nutrient of bacteria and is crucial for bacterial invasion. Reduced availability of iron by the host may cause significant stress for bacterial pathogens and is considered a signal that leads to significant alteration in virulence gene expression. However, the precise effect of iron-restriction on E. ictaluri protein abundance is unknown. The purpose of this study was to identify differentially abundant proteins of E. ictaluri during in vitro iron-restricted conditions. We applied two-dimensional difference in gel electrophoresis (2D-DIGE for determining differentially abundant proteins and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS for protein identification. Gene ontology and pathway-based functional modeling of differentially abundant proteins was also conducted. A total of 50 unique differentially abundant proteins at a minimum of 2-fold (p ≤ 0.05 difference in abundance due to iron-restriction were detected. The numbers of up- and down-regulated proteins were 37 and 13, respectively. We noted several proteins, including EsrB, LamB, MalM, MalE, FdaA, and TonB-dependent heme/hemoglobin receptor family proteins responded to iron restriction in E. ictaluri.

Dynamic protein S-palmitoylation mediates parasite life cycle progression and diverse mechanisms of virulence.

Science.gov (United States)

Brown, Robert W B; Sharma, Aabha I; Engman, David M

2017-04-01

Eukaryotic parasites possess complex life cycles and utilize an assortment of molecular mechanisms to overcome physical barriers, suppress and/or bypass the host immune response, including invading host cells where they can replicate in a protected intracellular niche. Protein S-palmitoylation is a dynamic post-translational modification in which the fatty acid palmitate is covalently linked to cysteine residues on proteins by the enzyme palmitoyl acyltransferase (PAT) and can be removed by lysosomal palmitoyl-protein thioesterase (PPT) or cytosolic acyl-protein thioesterase (APT). In addition to anchoring proteins to intracellular membranes, functions of dynamic palmitoylation include - targeting proteins to specific intracellular compartments via trafficking pathways, regulating the cycling of proteins between membranes, modulating protein function and regulating protein stability. Recent studies in the eukaryotic parasites - Plasmodium falciparum, Toxoplasma gondii, Trypanosoma brucei, Cryptococcus neoformans and Giardia lamblia - have identified large families of PATs and palmitoylated proteins. Many palmitoylated proteins are important for diverse aspects of pathogenesis, including differentiation into infective life cycle stages, biogenesis and tethering of secretory organelles, assembling the machinery powering motility and targeting virulence factors to the plasma membrane. This review aims to summarize our current knowledge of palmitoylation in eukaryotic parasites, highlighting five exemplary mechanisms of parasite virulence dependent on palmitoylation.

Quantifying protein dynamics in the ps–ns time regime by NMR relaxation

Energy Technology Data Exchange (ETDEWEB)

Hernández, Griselda; LeMaster, David M., E-mail: david.lemaster@health.ny.gov [University at Albany - SUNY, Wadsworth Center, New York State Department of Health and Department of Biomedical Sciences, School of Public Health (United States)

2016-11-15

Both {sup 15}N chemical shift anisotropy (CSA) and sufficiently rapid exchange linebroadening transitions exhibit relaxation contributions that are proportional to the square of the magnetic field. Deconvoluting these contributions is further complicated by residue-dependent variations in protein amide {sup 15}N CSA values which have proven difficult to accurately measure. Exploiting recently reported improvements for the implementation of T{sub 1} and T{sub 1ρ} experiments, field strength-dependent studies have been carried out on the B3 domain of protein G (GB3) as well as on the immunophilin FKBP12 and a H87V variant of that protein in which the major conformational exchange linebroadening transition is suppressed. By applying a zero frequency spectral density rescaling analysis to the relaxation data collected at magnetic fields from 500 to 900 MHz {sup 1}H, differential residue-specific {sup 15}N CSA values have been obtained for GB3 which correlate with those derived from solid state and liquid crystalline NMR measurements to a level similar to the correlation among those previously reported studies. Application of this analysis protocol to FKBP12 demonstrated an efficient quantitation of both weak exchange linebroadening contributions and differential residue-specific {sup 15}N CSA values. Experimental access to such differential residue-specific {sup 15}N CSA values should significantly facilitate more accurate comparisons with molecular dynamics simulations of protein motion that occurs within the timeframe of global molecular tumbling.

Exploring protein dynamics space: the dynasome as the missing link between protein structure and function.

Directory of Open Access Journals (Sweden)

Ulf Hensen

Full Text Available Proteins are usually described and classified according to amino acid sequence, structure or function. Here, we develop a minimally biased scheme to compare and classify proteins according to their internal mobility patterns. This approach is based on the notion that proteins not only fold into recurring structural motifs but might also be carrying out only a limited set of recurring mobility motifs. The complete set of these patterns, which we tentatively call the dynasome, spans a multi-dimensional space with axes, the dynasome descriptors, characterizing different aspects of protein dynamics. The unique dynamic fingerprint of each protein is represented as a vector in the dynasome space. The difference between any two vectors, consequently, gives a reliable measure of the difference between the corresponding protein dynamics. We characterize the properties of the dynasome by comparing the dynamics fingerprints obtained from molecular dynamics simulations of 112 proteins but our approach is, in principle, not restricted to any specific source of data of protein dynamics. We conclude that: 1. the dynasome consists of a continuum of proteins, rather than well separated classes. 2. For the majority of proteins we observe strong correlations between structure and dynamics. 3. Proteins with similar function carry out similar dynamics, which suggests a new method to improve protein function annotation based on protein dynamics.

Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis.

Science.gov (United States)

Chau, Johnnie; Kulnane, Laura Shapiro; Salz, Helen K

2012-06-12

Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3' untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior.

Automatic differentiation tools in the dynamic simulation of chemical engineering processes

Directory of Open Access Journals (Sweden)

Castro M.C.

2000-01-01

Full Text Available Automatic Differentiation is a relatively recent technique developed for the differentiation of functions applicable directly to the source code to compute the function written in standard programming languages. That technique permits the automatization of the differentiation step, crucial for dynamic simulation and optimization of processes. The values for the derivatives obtained with AD are exact (to roundoff. The theoretical exactness of the AD comes from the fact that it uses the same rules of differentiation as in differential calculus, but these rules are applied to an algorithmic specification of the function rather than to a formula. The main purpose of this contribution is to discuss the impact of Automatic Differentiation in the field of dynamic simulation of chemical engineering processes. The influence of the differentiation technique on the behavior of the integration code, the performance of the generated code and the incorporation of AD tools in consistent initialization tools are discussed from the viewpoint of dynamic simulation of typical models in chemical engineering.

Dissecting the calcium-induced differentiation of human primary keratinocytes stem cells by integrative and structural network analyses.

Directory of Open Access Journals (Sweden)

Kiana Toufighi

2015-05-01

Full Text Available The molecular details underlying the time-dependent assembly of protein complexes in cellular networks, such as those that occur during differentiation, are largely unexplored. Focusing on the calcium-induced differentiation of primary human keratinocytes as a model system for a major cellular reorganization process, we look at the expression of genes whose products are involved in manually-annotated protein complexes. Clustering analyses revealed only moderate co-expression of functionally related proteins during differentiation. However, when we looked at protein complexes, we found that the majority (55% are composed of non-dynamic and dynamic gene products ('di-chromatic', 19% are non-dynamic, and 26% only dynamic. Considering three-dimensional protein structures to predict steric interactions, we found that proteins encoded by dynamic genes frequently interact with a common non-dynamic protein in a mutually exclusive fashion. This suggests that during differentiation, complex assemblies may also change through variation in the abundance of proteins that compete for binding to common proteins as found in some cases for paralogous proteins. Considering the example of the TNF-α/NFκB signaling complex, we suggest that the same core complex can guide signals into diverse context-specific outputs by addition of time specific expressed subunits, while keeping other cellular functions constant. Thus, our analysis provides evidence that complex assembly with stable core components and competition could contribute to cell differentiation.

A stochastic differential equation analysis of cerebrospinal fluid dynamics.

Science.gov (United States)

Raman, Kalyan

2011-01-18

Clinical measurements of intracranial pressure (ICP) over time show fluctuations around the deterministic time path predicted by a classic mathematical model in hydrocephalus research. Thus an important issue in mathematical research on hydrocephalus remains unaddressed--modeling the effect of noise on CSF dynamics. Our objective is to mathematically model the noise in the data. The classic model relating the temporal evolution of ICP in pressure-volume studies to infusions is a nonlinear differential equation based on natural physical analogies between CSF dynamics and an electrical circuit. Brownian motion was incorporated into the differential equation describing CSF dynamics to obtain a nonlinear stochastic differential equation (SDE) that accommodates the fluctuations in ICP. The SDE is explicitly solved and the dynamic probabilities of exceeding critical levels of ICP under different clinical conditions are computed. A key finding is that the probabilities display strong threshold effects with respect to noise. Above the noise threshold, the probabilities are significantly influenced by the resistance to CSF outflow and the intensity of the noise. Fluctuations in the CSF formation rate increase fluctuations in the ICP and they should be minimized to lower the patient's risk. The nonlinear SDE provides a scientific methodology for dynamic risk management of patients. The dynamic output of the SDE matches the noisy ICP data generated by the actual intracranial dynamics of patients better than the classic model used in prior research.

Hydration dynamics near a model protein surface

International Nuclear Information System (INIS)

Russo, Daniela; Hura, Greg; Head-Gordon, Teresa

2003-01-01

The evolution of water dynamics from dilute to very high concentration solutions of a prototypical hydrophobic amino acid with its polar backbone, N-acetyl-leucine-methylamide (NALMA), is studied by quasi-elastic neutron scattering and molecular dynamics simulation for both the completely deuterated and completely hydrogenated leucine monomer. We observe several unexpected features in the dynamics of these biological solutions under ambient conditions. The NALMA dynamics shows evidence of de Gennes narrowing, an indication of coherent long timescale structural relaxation dynamics. The translational water dynamics are analyzed in a first approximation with a jump diffusion model. At the highest solute concentrations, the hydration water dynamics is significantly suppressed and characterized by a long residential time and a slow diffusion coefficient. The analysis of the more dilute concentration solutions takes into account the results of the 2.0M solution as a model of the first hydration shell. Subtracting the first hydration layer based on the 2.0M spectra, the translational diffusion dynamics is still suppressed, although the rotational relaxation time and residential time are converged to bulk-water values. Molecular dynamics analysis shows spatially heterogeneous dynamics at high concentration that becomes homogeneous at more dilute concentrations. We discuss the hydration dynamics results of this model protein system in the context of glassy systems, protein function, and protein-protein interfaces

3D Protein Dynamics in the Cell Nucleus.

Science.gov (United States)

Singh, Anand P; Galland, Rémi; Finch-Edmondson, Megan L; Grenci, Gianluca; Sibarita, Jean-Baptiste; Studer, Vincent; Viasnoff, Virgile; Saunders, Timothy E

2017-01-10

The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The Mathlet Toolkit: Creating Dynamic Applets for Differential Equations and Dynamical Systems

Science.gov (United States)

Decker, Robert

2011-01-01

Dynamic/interactive graphing applets can be used to supplement standard computer algebra systems such as Maple, Mathematica, Derive, or TI calculators, in courses such as Calculus, Differential Equations, and Dynamical Systems. The addition of this type of software can lead to discovery learning, with students developing their own conjectures, and…

Is Melanoma a stem cell tumor? Identification of neurogenic proteins in trans-differentiated cells

Directory of Open Access Journals (Sweden)

Chan Linda S

2005-03-01

Full Text Available Abstract Background Although several genes and proteins have been implicated in the development of melanomas, the molecular mechanisms involved in the development of these tumors are not well understood. To gain a better understanding of the relationship between the cell growth, tumorigenesis and differentiation, we have studied a highly malignant cat melanoma cell line that trans-differentiates into neuronal cells after exposure to a feline endogenous retrovirus RD114. Methods To define the repertoire of proteins responsible for the phenotypic differences between melanoma and its counterpart trans-differentiated neuronal cells we have applied proteomics technology and compared protein profiles of the two cell types and identified differentially expressed proteins by 2D-gel electrophoresis, image analyses and mass spectrometry. Results The melanoma and trans-differentiated neuronal cells could be distinguished by the presence of distinct sets of proteins in each. Although approximately 60–70% of the expressed proteins were shared between the two cell types, twelve proteins were induced de novo after infection of melanoma cells with RD114 virus in vitro. Expression of these proteins in trans-differentiated cells was significantly associated with concomitant down regulation of growth promoting proteins and up-regulation of neurogenic proteins (p = 95% proteins expressed in trans-differentiated cells could be associated with the development, differentiation and regulation of nervous system cells. Conclusion Our results indicate that the cat melanoma cells have the ability to differentiate into distinct neuronal cell types and they express proteins that are essential for self-renewal. Since melanocytes arise from the neural crest of the embryo, we conclude that this melanoma arose from embryonic precursor stem cells. This model system provides a unique opportunity to identify domains of interactions between the expressed proteins that halt the

Identification of differentially expressed proteins in response to Pb ...

African Journals Online (AJOL)

In response to Pb, a total of 76 proteins, out of the 95 differentially expressed proteins, were subjected to MALDI-TOF-MS Of these, 46 identities were identified by PMF and 19 identities were identified by microsequencing. Basic metabolisms such as photosynthesis, photorespiration and protein biosynthesis in C. roseus ...

A stochastic differential equation analysis of cerebrospinal fluid dynamics

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Raman Kalyan

2011-01-01

Full Text Available Abstract Background Clinical measurements of intracranial pressure (ICP over time show fluctuations around the deterministic time path predicted by a classic mathematical model in hydrocephalus research. Thus an important issue in mathematical research on hydrocephalus remains unaddressed--modeling the effect of noise on CSF dynamics. Our objective is to mathematically model the noise in the data. Methods The classic model relating the temporal evolution of ICP in pressure-volume studies to infusions is a nonlinear differential equation based on natural physical analogies between CSF dynamics and an electrical circuit. Brownian motion was incorporated into the differential equation describing CSF dynamics to obtain a nonlinear stochastic differential equation (SDE that accommodates the fluctuations in ICP. Results The SDE is explicitly solved and the dynamic probabilities of exceeding critical levels of ICP under different clinical conditions are computed. A key finding is that the probabilities display strong threshold effects with respect to noise. Above the noise threshold, the probabilities are significantly influenced by the resistance to CSF outflow and the intensity of the noise. Conclusions Fluctuations in the CSF formation rate increase fluctuations in the ICP and they should be minimized to lower the patient's risk. The nonlinear SDE provides a scientific methodology for dynamic risk management of patients. The dynamic output of the SDE matches the noisy ICP data generated by the actual intracranial dynamics of patients better than the classic model used in prior research.

Neisseria meningitidis rifampicin resistant strains: analysis of protein differentially expressed

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Schininà Maria

2010-09-01

Full Text Available Abstract Background Several mutations have been described as responsible for rifampicin resistance in Neisseria meningitidis. However, the intriguing question on why these strains are so rare remains open. The aim of this study was to investigate the protein content and to identify differential expression in specific proteins in two rifampicin resistant and one susceptible meningococci using two-dimensional electrophoresis (2-DE combined with mass spectrometry. Results In our experimental conditions, able to resolve soluble proteins with an isoelectric point between 4 and 7, twenty-three proteins have been found differentially expressed in the two resistant strains compared to the susceptible. Some of them, involved in the main metabolic pathways, showed an increased expression, mainly in the catabolism of pyruvate and in the tricarboxylic acid cycle. A decreased expression of proteins belonging to gene regulation and to those involved in the folding of polypeptides has also been observed. 2-DE analysis showed the presence of four proteins displaying a shift in their isoelectric point in both resistant strains, confirmed by the presence of amino acid changes in the sequence analysis, absent in the susceptible. Conclusions The analysis of differentially expressed proteins suggests that an intricate series of events occurs in N. meningitidis rifampicin resistant strains and the results here reported may be considered a starting point in understanding their decreased invasion capacity. In fact, they support the hypothesis that the presence of more than one protein differentially expressed, having a role in the metabolism of the meningococcus, influences its ability to infect and to spread in the population. Different reports have described and discussed how a drug resistant pathogen shows a high biological cost for survival and that may also explain why, for some pathogens, the rate of resistant organisms is relatively low considering the

Simultaneous determination of protein structure and dynamics

DEFF Research Database (Denmark)

Lindorff-Larsen, Kresten; Best, Robert B.; DePristo, M. A.

2005-01-01

at the atomic level about the structural and dynamical features of proteins-with the ability of molecular dynamics simulations to explore a wide range of protein conformations. We illustrate the method for human ubiquitin in solution and find that there is considerable conformational heterogeneity throughout......We present a protocol for the experimental determination of ensembles of protein conformations that represent simultaneously the native structure and its associated dynamics. The procedure combines the strengths of nuclear magnetic resonance spectroscopy-for obtaining experimental information...... the protein structure. The interior atoms of the protein are tightly packed in each individual conformation that contributes to the ensemble but their overall behaviour can be described as having a significant degree of liquid-like character. The protocol is completely general and should lead to significant...

FSHD myoblasts fail to downregulate intermediate filament protein vimentin during myogenic differentiation.

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Lipinski M.

2011-10-01

Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is an autosomal dominant hereditary neuromuscular disorder. The clinical features of FSHD include weakness of the facial and shoulder girdle muscles followed by wasting of skeletal muscles of the pelvic girdle and lower extremities. Although FSHD myoblasts grown in vitro can be induced to differentiate into myotubes by serum starvation, the resulting FSHD myotubes have been shown previously to be morphologically abnormal. Aim. In order to find the cause of morphological anomalies of FSHD myotubes we compared in vitro myogenic differentiation of normal and FSHD myoblasts at the protein level. Methods. We induced myogenic differentiation of normal and FSHD myoblasts by serum starvation. We then compared protein extracts from proliferating myoblasts and differentiated myotubes using SDS-PAGE followed by mass spectrometry identification of differentially expressed proteins. Results. We demonstrated that the expression of vimentin was elevated at the protein and mRNA levels in FSHD myotubes as compared to normal myotubes. Conclusions. We demonstrate for the first time that in contrast to normal myoblasts, FSHD myoblasts fail to downregulate vimentin after induction of in vitro myogenic differentiation. We suggest that vimentin could be an easily detectable marker of FSHD myotubes

Exploration of the dynamic properties of protein complexes predicted from spatially constrained protein-protein interaction networks.

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Eric A Yen

2014-05-01

Full Text Available Protein complexes are not static, but rather highly dynamic with subunits that undergo 1-dimensional diffusion with respect to each other. Interactions within protein complexes are modulated through regulatory inputs that alter interactions and introduce new components and deplete existing components through exchange. While it is clear that the structure and function of any given protein complex is coupled to its dynamical properties, it remains a challenge to predict the possible conformations that complexes can adopt. Protein-fragment Complementation Assays detect physical interactions between protein pairs constrained to ≤8 nm from each other in living cells. This method has been used to build networks composed of 1000s of pair-wise interactions. Significantly, these networks contain a wealth of dynamic information, as the assay is fully reversible and the proteins are expressed in their natural context. In this study, we describe a method that extracts this valuable information in the form of predicted conformations, allowing the user to explore the conformational landscape, to search for structures that correlate with an activity state, and estimate the abundance of conformations in the living cell. The generator is based on a Markov Chain Monte Carlo simulation that uses the interaction dataset as input and is constrained by the physical resolution of the assay. We applied this method to an 18-member protein complex composed of the seven core proteins of the budding yeast Arp2/3 complex and 11 associated regulators and effector proteins. We generated 20,480 output structures and identified conformational states using principle component analysis. We interrogated the conformation landscape and found evidence of symmetry breaking, a mixture of likely active and inactive conformational states and dynamic exchange of the core protein Arc15 between core and regulatory components. Our method provides a novel tool for prediction and

Chromatin plasticity as a differentiation index during muscle differentiation of C2C12 myoblasts

International Nuclear Information System (INIS)

Watanabe, Tomonobu M.; Higuchi, Sayaka; Kawauchi, Keiko; Tsukasaki, Yoshikazu; Ichimura, Taro; Fujita, Hideaki

2012-01-01

Highlights: ► Change in the epigenetic landscape during myogenesis was optically investigated. ► Mobility of nuclear proteins was used to state the epigenetic status of the cell. ► Mobility of nuclear proteins decreased as myogenesis progressed in C2C12. ► Differentiation state diagram was developed using parameters obtained. -- Abstract: Skeletal muscle undergoes complicated differentiation steps that include cell-cycle arrest, cell fusion, and maturation, which are controlled through sequential expression of transcription factors. During muscle differentiation, remodeling of the epigenetic landscape is also known to take place on a large scale, determining cell fate. In an attempt to determine the extent of epigenetic remodeling during muscle differentiation, we characterized the plasticity of the chromatin structure using C2C12 myoblasts. Differentiation of C2C12 cells was induced by lowering the serum concentration after they had reached full confluence, resulting in the formation of multi-nucleated myotubes. Upon induction of differentiation, the nucleus size decreased whereas the aspect ratio increased, indicating the presence of force on the nucleus during differentiation. Movement of the nucleus was also suppressed when differentiation was induced, indicating that the plasticity of chromatin changed upon differentiation. To evaluate the histone dynamics during differentiation, FRAP experiment was performed, which showed an increase in the immobile fraction of histone proteins when differentiation was induced. To further evaluate the change in the histone dynamics during differentiation, FCS was performed, which showed a decrease in histone mobility on differentiation. We here show that the plasticity of chromatin decreases upon differentiation, which takes place in a stepwise manner, and that it can be used as an index for the differentiation stage during myogenesis using the state diagram developed with the parameters obtained in this study.

Fos and jun proteins are specifically expressed during differentiation of human keratinocytes.

Science.gov (United States)

Mehic, Denis; Bakiri, Latifa; Ghannadan, Minoo; Wagner, Erwin F; Tschachler, Erwin

2005-01-01

Activator protein 1 (AP-1) proteins play key roles in the regulation of cell proliferation and differentiation. In this study we investigated the expression of Fos and Jun proteins in different models of terminal differentiation of human keratinocytes and in skin from psoriasis patients. All Jun and Fos proteins, with the exception of FosB, were efficiently expressed in keratinocytes in monolayer cultures. In contrast, in normal epidermis as well as in organotypic epidermal cultures, the expression pattern of AP-1 proteins was dependent on the differentiation stage. Fos proteins were readily detected in nuclei of keratinocytes of basal and suprabasal layers. JunB and JunD were expressed in all layers of normal epidermis. Interestingly, expression of c-Jun started suprabasally, then disappeared and became detectable again in distinct cells of the outermost granular layer directly at the transition zone to the stratum corneum. In psoriatic epidermis, c-Jun expression was prominent in both hyperproliferating basal and suprabasal keratinocytes, whereas c-Fos expression was unchanged. These data indicate that AP-1 proteins are expressed in a highly specific manner during terminal differentiation of keratinocytes and that the enhanced expression of c-Jun in basal and suprabasal keratinocytes might contribute to the pathogenesis of psoriasis.

On the dynamical incompleteness of the Protein Data Bank.

Science.gov (United States)

Marino-Buslje, Cristina; Monzon, Alexander Miguel; Zea, Diego Javier; Fornasari, María Silvina; Parisi, Gustavo

2017-08-02

Major scientific challenges that are beyond the capability of individuals need to be addressed by multi-disciplinary and multi-institutional consortia. Examples of these endeavours include the Human Genome Project, and more recently, the Structural Genomics (SG) initiative. The SG initiative pursues the expansion of structural coverage to include at least one structural representative for each protein family to derive the remaining structures using homology modelling. However, biological function is inherently connected with protein dynamics that can be studied by knowing different structures of the same protein. This ensemble of structures provides snapshots of protein conformational diversity under native conditions. Thus, sequence redundancy in the Protein Data Bank (PDB) (i.e. crystallization of the same protein under different conditions) is therefore an essential input contributing to experimentally based studies of protein dynamics and providing insights into protein function. In this work, we show that sequence redundancy, a key concept for exploring protein dynamics, is highly biased and fundamentally incomplete in the PDB. Additionally, our results show that dynamical behaviour of proteins cannot be inferred using homologous proteins. Minor to moderate changes in sequence can produce great differences in dynamical behaviour. Nonetheless, the structural and dynamical incompleteness of the PDB is apparently unrelated concepts in SG. While the first could be reversed by promoting the extension of the structural coverage, we would like to emphasize that further focused efforts will be needed to amend the incompleteness of the PDB in terms of dynamical information content, essential to fully understand protein function. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Revealing Pathway Dynamics in Heart Diseases by Analyzing Multiple Differential Networks.

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Xiaoke Ma

2015-06-01

Full Text Available Development of heart diseases is driven by dynamic changes in both the activity and connectivity of gene pathways. Understanding these dynamic events is critical for understanding pathogenic mechanisms and development of effective treatment. Currently, there is a lack of computational methods that enable analysis of multiple gene networks, each of which exhibits differential activity compared to the network of the baseline/healthy condition. We describe the iMDM algorithm to identify both unique and shared gene modules across multiple differential co-expression networks, termed M-DMs (multiple differential modules. We applied iMDM to a time-course RNA-Seq dataset generated using a murine heart failure model generated on two genotypes. We showed that iMDM achieves higher accuracy in inferring gene modules compared to using single or multiple co-expression networks. We found that condition-specific M-DMs exhibit differential activities, mediate different biological processes, and are enriched for genes with known cardiovascular phenotypes. By analyzing M-DMs that are present in multiple conditions, we revealed dynamic changes in pathway activity and connectivity across heart failure conditions. We further showed that module dynamics were correlated with the dynamics of disease phenotypes during the development of heart failure. Thus, pathway dynamics is a powerful measure for understanding pathogenesis. iMDM provides a principled way to dissect the dynamics of gene pathways and its relationship to the dynamics of disease phenotype. With the exponential growth of omics data, our method can aid in generating systems-level insights into disease progression.

Differential equations, dynamical systems, and an introduction to chaos

CERN Document Server

Smale, Stephen; Devaney, Robert L

2003-01-01

Thirty years in the making, this revised text by three of the world''s leading mathematicians covers the dynamical aspects of ordinary differential equations. it explores the relations between dynamical systems and certain fields outside pure mathematics, and has become the standard textbook for graduate courses in this area. The Second Edition now brings students to the brink of contemporary research, starting from a background that includes only calculus and elementary linear algebra.The authors are tops in the field of advanced mathematics, including Steve Smale who is a recipient of the Field''s Medal for his work in dynamical systems.* Developed by award-winning researchers and authors* Provides a rigorous yet accessible introduction to differential equations and dynamical systems* Includes bifurcation theory throughout* Contains numerous explorations for students to embark uponNEW IN THIS EDITION* New contemporary material and updated applications* Revisions throughout the text, including simplification...

Dynamic protein assembly by programmable DNA strand displacement

Science.gov (United States)

Chen, Rebecca P.; Blackstock, Daniel; Sun, Qing; Chen, Wilfred

2018-03-01

Inspired by the remarkable ability of natural protein switches to sense and respond to a wide range of environmental queues, here we report a strategy to engineer synthetic protein switches by using DNA strand displacement to dynamically organize proteins with highly diverse and complex logic gate architectures. We show that DNA strand displacement can be used to dynamically control the spatial proximity and the corresponding fluorescence resonance energy transfer between two fluorescent proteins. Performing Boolean logic operations enabled the explicit control of protein proximity using multi-input, reversible and amplification architectures. We further demonstrate the power of this technology beyond sensing by achieving dynamic control of an enzyme cascade. Finally, we establish the utility of the approach as a synthetic computing platform that drives the dynamic reconstitution of a split enzyme for targeted prodrug activation based on the sensing of cancer-specific miRNAs.

Water dynamics clue to key residues in protein folding

International Nuclear Information System (INIS)

Gao, Meng; Zhu, Huaiqiu; Yao, Xin-Qiu; She, Zhen-Su

2010-01-01

A computational method independent of experimental protein structure information is proposed to recognize key residues in protein folding, from the study of hydration water dynamics. Based on all-atom molecular dynamics simulation, two key residues are recognized with distinct water dynamical behavior in a folding process of the Trp-cage protein. The identified key residues are shown to play an essential role in both 3D structure and hydrophobic-induced collapse. With observations on hydration water dynamics around key residues, a dynamical pathway of folding can be interpreted.

Inhibition of protein kinase C induces differentiation in Neuro-2a cells

International Nuclear Information System (INIS)

Minana, M.D.; Felipo, V.; Grisolia, S.

1990-01-01

1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinase C, induced neuritogenesis in Neuro-2a cells, whereas N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), which inhibits more efficiently cAMP- and cGMP-dependent protein kinases, did not. The effect, noticeable after 3 hr, was maximum (13-fold increase at 500 μM H7) between 1 and 3 days and was maintained over 2 months. In controls, 90% of the cells were undifferentiated, whereas after 3 hr with 500 μM H7 only 25% of the cells remained undifferentiated. DNA synthesis decreased as the number of differentiated cells increased. Differentiation is also functional since acetylcholinesterase activity increased ∼7-fold after 48 hr with 500 μM H7. Phorbol 12-myristate 13-acetate, a specific activator of protein kinase C, prevented or reversed the induction of neuritogenesis and the inhibition of DNA synthesis by H7. There is a good correlation between the level of protein kinase C and the percentage of differentiated cells. The results indicate that protein kinase C may play a key role in the control of differentiation of neural cells. Some possible clinical implications are briefly discussed

Modelling Protein Dynamics on the Microsecond Time Scale

DEFF Research Database (Denmark)

Siuda, Iwona Anna

Recent years have shown an increase in coarse-grained (CG) molecular dynamics simulations, providing structural and dynamic details of large proteins and enabling studies of self-assembly of biological materials. It is not easy to acquire such data experimentally, and access is also still limited...... in atomistic simulations. During her PhD studies, Iwona Siuda used MARTINI CG models to study the dynamics of different globular and membrane proteins. In several cases, the MARTINI model was sufficient to study conformational changes of small, purely alpha-helical proteins. However, in studies of larger......ELNEDIN was therefore proposed as part of the work. Iwona Siuda’s results from the CG simulations had biological implications that provide insights into possible mechanisms of the periplasmic leucine-binding protein, the sarco(endo)plasmic reticulum calcium pump, and several proteins from the saposin-like proteins...

Stabilities and Dynamics of Protein Folding Nuclei by Molecular Dynamics Simulation

Science.gov (United States)

Song, Yong-Shun; Zhou, Xin; Zheng, Wei-Mou; Wang, Yan-Ting

2017-07-01

To understand how the stabilities of key nuclei fragments affect protein folding dynamics, we simulate by molecular dynamics (MD) simulation in aqueous solution four fragments cut out of a protein G, including one α-helix (seqB: KVFKQYAN), two β-turns (seqA: LNGKTLKG and seqC: YDDATKTF), and one β-strand (seqD: DGEWTYDD). The Markov State Model clustering method combined with the coarse-grained conformation letters method are employed to analyze the data sampled from 2-μs equilibrium MD simulation trajectories. We find that seqA and seqB have more stable structures than their native structures which become metastable when cut out of the protein structure. As expected, seqD alone is flexible and does not have a stable structure. Throughout our simulations, the native structure of seqC is stable but cannot be reached if starting from a structure other than the native one, implying a funnel-shape free energy landscape of seqC in aqueous solution. All the above results suggest that different nuclei have different formation dynamics during protein folding, which may have a major contribution to the hierarchy of protein folding dynamics. Supported by the National Basic Research Program of China under Grant No. 2013CB932804, the National Natural Science Foundation of China under Grant No. 11421063, and the CAS Biophysics Interdisciplinary Innovation Team Project

Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

Energy Technology Data Exchange (ETDEWEB)

Walsh, Erica M. [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Niu, MengMeng; Bergholz, Johann [Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China); Jim Xiao, Zhi-Xiong, E-mail: jxiao@bu.edu [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China)

2015-05-29

The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation. - Highlights: • Nutlin-3 inhibits myoblast proliferation and prevents differentiation into myotubes. • Nutlin-3 increases MDM2 expression and down-regulates Rb protein levels. • This study has implication in nutlin-3 treatment of rhabdomyosarcomas.

Structure and dynamics of photosynthetic proteins studied by neutron scattering and molecular dynamic simulation

International Nuclear Information System (INIS)

Dellerue, Serge

2000-01-01

Understand the structure-dynamics-function relation in the case of proteins is essential. But few experimental techniques allow to have access to knowledge of fast internal movements of biological macromolecules. With the neutron scattering method, it has been possible to study the reorientation dynamics of side chains and of polypeptide skeleton for two proteins in terms of water or detergent and of temperature. With the use of the molecular dynamics method, essential for completing and interpreting the experimental data, it has been possible to assess the different contributions of the whole structure of proteins to the overall dynamics. It has been shown that the polypeptide skeleton presents an energy relaxation comparable to those of the side chains. Moreover, it has been explained that the protein dynamics can only be understood in terms of relaxation time distribution. (author) [fr

Dynamic changes in Id3 and E-protein activity orchestrate germinal center and plasma cell development

Science.gov (United States)

Gloury, Renee; Zotos, Dimitra; Zuidscherwoude, Malou; Masson, Frederick; Liao, Yang; Hasbold, Jhaguaral; Corcoran, Lynn M.; Hodgkin, Phil D.; Belz, Gabrielle T.; Shi, Wei; Nutt, Stephen L.; Tarlinton, David M.

2016-01-01

The generation of high-affinity antibodies requires germinal center (GC) development and differentiation of long-lived plasma cells in a multilayered process that is tightly controlled by the activity of multiple transcription factors. Here, we reveal a new layer of complexity by demonstrating that dynamic changes in Id3 and E-protein activity govern both GC and plasma cell differentiation. We show that down-regulation of Id3 in B cells is essential for releasing E2A and E2-2, which in a redundant manner are required for antigen-induced B cell differentiation. We demonstrate that this pathway controls the expression of multiple key factors, including Blimp1, Xbp1, and CXCR4, and is therefore critical for establishing the transcriptional network that controls GC B cell and plasma cell differentiation. PMID:27217539

Protein electron transfer: is biology (thermo)dynamic?

International Nuclear Information System (INIS)

Matyushov, Dmitry V

2015-01-01

Simple physical mechanisms are behind the flow of energy in all forms of life. Energy comes to living systems through electrons occupying high-energy states, either from food (respiratory chains) or from light (photosynthesis). This energy is transformed into the cross-membrane proton-motive force that eventually drives all biochemistry of the cell. Life’s ability to transfer electrons over large distances with nearly zero loss of free energy is puzzling and has not been accomplished in synthetic systems. The focus of this review is on how this energetic efficiency is realized. General physical mechanisms and interactions that allow proteins to fold into compact water-soluble structures are also responsible for a rugged landscape of energy states and a broad distribution of relaxation times. Specific to a protein as a fluctuating thermal bath is the protein-water interface, which is heterogeneous both dynamically and structurally. The spectrum of interfacial fluctuations is a consequence of protein’s elastic flexibility combined with a high density of surface charges polarizing water dipoles into surface nanodomains. Electrostatics is critical to the protein function and the relevant questions are: (i) What is the spectrum of interfacial electrostatic fluctuations? (ii) Does the interfacial biological water produce electrostatic signatures specific to proteins? (iii) How is protein-mediated chemistry affected by electrostatics? These questions connect the fluctuation spectrum to the dynamical control of chemical reactivity, i.e. the dependence of the activation free energy of the reaction on the dynamics of the bath. Ergodicity is often broken in protein-driven reactions and thermodynamic free energies become irrelevant. Continuous ergodicity breaking in a dense spectrum of relaxation times requires using dynamically restricted ensembles to calculate statistical averages. When applied to the calculation of the rates, this formalism leads to the nonergodic

Proteins with Novel Structure, Function and Dynamics

Science.gov (United States)

Pohorille, Andrew

2014-01-01

Recently, a small enzyme that ligates two RNA fragments with the rate of 10(exp 6) above background was evolved in vitro (Seelig and Szostak, Nature 448:828-831, 2007). This enzyme does not resemble any contemporary protein (Chao et al., Nature Chem. Biol. 9:81-83, 2013). It consists of a dynamic, catalytic loop, a small, rigid core containing two zinc ions coordinated by neighboring amino acids, and two highly flexible tails that might be unimportant for protein function. In contrast to other proteins, this enzyme does not contain ordered secondary structure elements, such as alpha-helix or beta-sheet. The loop is kept together by just two interactions of a charged residue and a histidine with a zinc ion, which they coordinate on the opposite side of the loop. Such structure appears to be very fragile. Surprisingly, computer simulations indicate otherwise. As the coordinating, charged residue is mutated to alanine, another, nearby charged residue takes its place, thus keeping the structure nearly intact. If this residue is also substituted by alanine a salt bridge involving two other, charged residues on the opposite sides of the loop keeps the loop in place. These adjustments are facilitated by high flexibility of the protein. Computational predictions have been confirmed experimentally, as both mutants retain full activity and overall structure. These results challenge our notions about what is required for protein activity and about the relationship between protein dynamics, stability and robustness. We hypothesize that small, highly dynamic proteins could be both active and fault tolerant in ways that many other proteins are not, i.e. they can adjust to retain their structure and activity even if subjected to mutations in structurally critical regions. This opens the doors for designing proteins with novel functions, structures and dynamics that have not been yet considered.

The dynamics of crude oil price differentials

International Nuclear Information System (INIS)

Fattouh, Bassam

2010-01-01

Crude oil price differentials are modelled as a two-regime threshold autoregressive (TAR) process using the method proposed by Caner and Hansen [Caner, M., Hansen, B.E. Threshold autoregression with a unit root. Econometrica 2001; 69; 1555-1596.]. While standard unit root tests suggest that the prices of crude oil of different varieties move closely together such that their price differential is stationary, the TAR results indicate strong evidence of threshold effects in the adjustment process to the long-run equilibrium. These findings suggest that crude oil prices are linked and thus at the very general level, the oil market is 'one great pool' (Adelman, M.A. International oil agreements. The Energy Journal 1984; 5; 1-9.). However, differences in the dynamics of adjustment suggest that within this one pool, oil markets are not necessarily integrated in every time period and hence the dynamics of crude oil price differentials may not follow a stationary process at all times. Although the development of a liquid futures market around the crude oil benchmarks has helped make some distant markets more unified, arbitrage is not costless or risk-free and temporary breakdowns in the benchmarks can lead to decoupling of crude oil prices. (author)

The Cx43-like connexin protein Cx40.8 is differentially localized during fin ontogeny and fin regeneration.

Directory of Open Access Journals (Sweden)

Sarah V Gerhart

Full Text Available Connexins (Cx are the subunits of gap junctions, membraneous protein channels that permit the exchange of small molecules between adjacent cells. Cx43 is required for cell proliferation in the zebrafish caudal fin. Previously, we found that a Cx43-like connexin, cx40.8, is co-expressed with cx43 in the population of proliferating cells during fin regeneration. Here we demonstrate that Cx40.8 exhibits novel differential subcellular localization in vivo, depending on the growth status of the fin. During fin ontogeny, Cx40.8 is found at the plasma membrane, but Cx40.8 is retained in the Golgi apparatus during regeneration. We next identified a 30 amino acid domain of Cx40.8 responsible for its dynamic localization. One possible explanation for the differential localization is that Cx40.8 contributes to the regulation of Cx43 in vivo, perhaps modifying channel activity during ontogenetic growth. However, we find that the voltage-gating properties of Cx40.8 are similar to Cx43. Together our findings reveal that Cx40.8 exhibits differential subcellular localization in vivo, dependent on a discrete domain in its carboxy terminus. We suggest that the dynamic localization of Cx40.8 differentially influences Cx43-dependent cell proliferation during ontogeny and regeneration.

The dynamic multisite interactions between two intrinsically disordered proteins

KAUST Repository

Wu, Shaowen

2017-05-11

Protein interactions involving intrinsically disordered proteins (IDPs) comprise a variety of binding modes, from the well characterized folding upon binding to dynamic fuzzy complex. To date, most studies concern the binding of an IDP to a structured protein, while the Interaction between two IDPs is poorly understood. In this study, we combined NMR, smFRET, and molecular dynamics (MD) simulation to characterize the interaction between two IDPs, the C-terminal domain (CTD) of protein 4.1G and the nuclear mitotic apparatus (NuMA) protein. It is revealed that CTD and NuMA form a fuzzy complex with remaining structural disorder. Multiple binding sites on both proteins were identified by MD and mutagenesis studies. Our study provides an atomic scenario in which two IDPs bearing multiple binding sites interact with each other in dynamic equilibrium. The combined approach employed here could be widely applicable for investigating IDPs and their dynamic interactions.

Constraint theory and hierarchical protein dynamics

International Nuclear Information System (INIS)

Phillips, J C

2004-01-01

The complexity and functionality of proteins requires that they occupy an exponentially small fraction of configuration space (perhaps 10 -300 ). How did evolution manage to create such unlikely objects? Thorpe has solved the static half of this problem (known in protein chemistry as Levinthal's paradox) by observing that for stress-free chain segments the complexity of optimally constrained elastic networks scales not with expN (where N ∼ 100-1000 is the number of amino acids in a protein), but only with N. Newman's results for diffusion in N-dimensional spaces provide suggestive insights into the dynamical half of the problem. He showed that the distribution of residence (or pausing) time between sign reversals changes qualitatively at N ∼40. The overall sign of a protein can be defined in terms of a product of curvature and hydrophobic(philic) character over all amino acid residues. This construction agrees with the sizes of the smallest known proteins and prions, and it suggests a universal clock for protein molecular dynamics simulations

Constraint theory and hierarchical protein dynamics

Energy Technology Data Exchange (ETDEWEB)

Phillips, J C [Department of Physics and Astronomy, Rutgers University, Piscataway, NJ 08854-8019 (United States)

2004-11-10

The complexity and functionality of proteins requires that they occupy an exponentially small fraction of configuration space (perhaps 10{sup -300}). How did evolution manage to create such unlikely objects? Thorpe has solved the static half of this problem (known in protein chemistry as Levinthal's paradox) by observing that for stress-free chain segments the complexity of optimally constrained elastic networks scales not with expN (where N {approx} 100-1000 is the number of amino acids in a protein), but only with N. Newman's results for diffusion in N-dimensional spaces provide suggestive insights into the dynamical half of the problem. He showed that the distribution of residence (or pausing) time between sign reversals changes qualitatively at N {approx}40. The overall sign of a protein can be defined in terms of a product of curvature and hydrophobic(philic) character over all amino acid residues. This construction agrees with the sizes of the smallest known proteins and prions, and it suggests a universal clock for protein molecular dynamics simulations.

Protein dynamics in individual human cells: experiment and theory.

Directory of Open Access Journals (Sweden)

Ariel Aharon Cohen

Full Text Available A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle-dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell-cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell-cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells.

ProteinAC: a frequency domain technique for analyzing protein dynamics

Science.gov (United States)

Bozkurt Varolgunes, Yasemin; Demir, Alper

2018-03-01

It is widely believed that the interactions of proteins with ligands and other proteins are determined by their dynamic characteristics as opposed to only static, time-invariant processes. We propose a novel computational technique, called ProteinAC (PAC), that can be used to analyze small scale functional protein motions as well as interactions with ligands directly in the frequency domain. PAC was inspired by a frequency domain analysis technique that is widely used in electronic circuit design, and can be applied to both coarse-grained and all-atom models. It can be considered as a generalization of previously proposed static perturbation-response methods, where the frequency of the perturbation becomes the key. We discuss the precise relationship of PAC to static perturbation-response schemes. We show that the frequency of the perturbation may be an important factor in protein dynamics. Perturbations at different frequencies may result in completely different response behavior while magnitude and direction are kept constant. Furthermore, we introduce several novel frequency dependent metrics that can be computed via PAC in order to characterize response behavior. We present results for the ferric binding protein that demonstrate the potential utility of the proposed techniques.

Functional dynamics of cell surface membrane proteins.

Science.gov (United States)

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.

Phosphorylation dynamics during early differentiation of human embryonic stem cells

NARCIS (Netherlands)

van Hoof, D.; Munoz, J.; Braam, S.R.; Pinkse, M.W.H.; Linding, R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

2009-01-01

Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during

Novel function of the retinoblastoma protein in fat: regulation of white versus brown adipocyte differentiation

DEFF Research Database (Denmark)

Hansen, Jacob B; te Riele, Hein; Kristiansen, Karsten

2004-01-01

the major energy store and brown adipocytes being potent energy-dissipaters through thermogenesis. Yet, little is known about factors differentially regulating the formation of white and brown fat cells. Members of the retinoblastoma protein family (pRB, p107, p130) have been implicated in the regulation...... of adipocyte differentiation, and expression and phosphorylation of the three retinoblastoma family proteins oscillate in a characteristic manner during differentiation of the white preadipocyte cell line 3T3-L1. We have recently demonstrated a surprising function of the retinoblastoma protein...... in the regulation of white versus brown adipocyte differentiation in vitro and possibly in vivo. Here we summarize the current knowledge on the retinoblastoma protein in fat cells, with particular emphasis on its potential role in adipocyte lineage commitment and differentiation....

Lineage-specific interface proteins match up the cell cycle and differentiation in embryo stem cells

DEFF Research Database (Denmark)

Re, Angela; Workman, Christopher; Waldron, Levi

2014-01-01

The shortage of molecular information on cell cycle changes along embryonic stem cell (ESC) differentiation prompts an in silico approach, which may provide a novel way to identify candidate genes or mechanisms acting in coordinating the two programs. We analyzed germ layer specific gene expression...... changes during the cell cycle and ESC differentiation by combining four human cell cycle transcriptome profiles with thirteen in vitro human ESC differentiation studies. To detect cross-talk mechanisms we then integrated the transcriptome data that displayed differential regulation with protein...... interaction data. A new class of non-transcriptionally regulated genes was identified, encoding proteins which interact systematically with proteins corresponding to genes regulated during the cell cycle or cell differentiation, and which therefore can be seen as interface proteins coordinating the two...

In silico modelling and validation of differential expressed proteins in lung cancer

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Bhagavathi S

2012-05-01

Full Text Available Objective: The present study aims predict the three dimensional structure of three major proteins responsible for causing Lung cancer. Methods: These are the differentially expressed proteins in lung cancer dataset. Initially, the structural template for these proteins is identified from structural database using homology search and perform homology modelling approach to predict its native 3D structure. Three-dimensional model obtained was validated using Ramachandran plot analysis to find the reliability of the model. Results: Four proteins were differentially expressed and were significant proteins in causing lung cancer. Among the four proteins, Matrixmetallo proteinase (P39900 had a known 3D structure and hence was not considered for modelling. The remaining proteins Polo like kinase I Q58A51, Trophinin B1AKF1, Thrombomodulin P07204 were modelled and validated. Conclusions: The three dimensional structure of proteins provides insights about the functional aspect and regulatory aspect of the protein. Thus, this study will be a breakthrough for further lung cancer related studies.

Differential Editosome Protein Function between Life Cycle Stages of Trypanosoma brucei.

Science.gov (United States)

McDermott, Suzanne M; Guo, Xuemin; Carnes, Jason; Stuart, Kenneth

2015-10-09

Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei. The mRNAs are differentially edited in bloodstream form (BF) and procyclic form (PF) life cycle stages, and this correlates with the differential utilization of glycolysis and oxidative phosphorylation between the stages. The mechanism that controls this differential editing is unknown. Editing is catalyzed by multiprotein ∼20S editosomes that contain endonuclease, 3'-terminal uridylyltransferase, exonuclease, and ligase activities. These editosomes also contain KREPB5 and KREPA3 proteins, which have no functional catalytic motifs, but they are essential for parasite viability, editing, and editosome integrity in BF cells. We show here that repression of KREPB5 or KREPA3 is also lethal in PF, but the effects on editosome structure differ from those in BF. In addition, we found that point mutations in KREPB5 or KREPA3 differentially affect cell growth, editosome integrity, and RNA editing between BF and PF stages. These results indicate that the functions of KREPB5 and KREPA3 editosome proteins are adjusted between the life cycle stages. This implies that these proteins are involved in the processes that control differential editing and that the 20S editosomes differ between the life cycle stages. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Inactivation of Tor proteins affects the dynamics of endocytic proteins ...

Indian Academy of Sciences (India)

Tor2 is an activator of the Rom2/Rho1 pathway that regulates -factor internalization. Since the recruitment of endocytic proteins such as actin-binding proteins and the amphiphysins precedes the internalization of -factor, we hypothesized that loss of Tor function leads to an alteration in the dynamics of the endocytic ...

Dynamics of DNA conformations and DNA-protein interaction

DEFF Research Database (Denmark)

Metzler, R.; Ambjörnsson, T.; Lomholt, Michael Andersen

2005-01-01

Optical tweezers, atomic force microscopes, patch clamping, or fluorescence techniques make it possible to study both the equilibrium conformations and dynamics of single DNA molecules as well as their interaction with binding proteins. In this paper we address the dynamics of local DNA...... denaturation (bubble breathing), deriving its dynamic response to external physical parameters and the DNA sequence in terms of the bubble relaxation time spectrum and the autocorrelation function of bubble breathing. The interaction with binding proteins that selectively bind to the DNA single strand exposed...... in a denaturation bubble are shown to involve an interesting competition of time scales, varying between kinetic blocking of protein binding up to full binding protein-induced denaturation of the DNA. We will also address the potential to use DNA physics for the design of nanosensors. Finally, we report recent...

PDB2CD visualises dynamics within protein structures.

Science.gov (United States)

Janes, Robert W

2017-10-01

Proteins tend to have defined conformations, a key factor in enabling their function. Atomic resolution structures of proteins are predominantly obtained by either solution nuclear magnetic resonance (NMR) or crystal structure methods. However, when considering a protein whose structure has been determined by both these approaches, on many occasions, the resultant conformations are subtly different, as illustrated by the examples in this study. The solution NMR approach invariably results in a cluster of structures whose conformations satisfy the distance boundaries imposed by the data collected; it might be argued that this is evidence of the dynamics of proteins when in solution. In crystal structures, the proteins are often in an energy minimum state which can result in an increase in the extent of regular secondary structure present relative to the solution state depicted by NMR, because the more dynamic ends of alpha helices and beta strands can become ordered at the lower temperatures. This study examines a novel way to display the differences in conformations within an NMR ensemble and between these and a crystal structure of a protein. Circular dichroism (CD) spectroscopy can be used to characterise protein structures in solution. Using the new bioinformatics tool, PDB2CD, which generates CD spectra from atomic resolution protein structures, the differences between, and possible dynamic range of, conformations adopted by a protein can be visualised.

A hybrid approach to protein differential expression in mass spectrometry-based proteomics

KAUST Repository

Wang, X.

2012-04-19

MOTIVATION: Quantitative mass spectrometry-based proteomics involves statistical inference on protein abundance, based on the intensities of each protein\\'s associated spectral peaks. However, typical MS-based proteomics datasets have substantial proportions of missing observations, due at least in part to censoring of low intensities. This complicates intensity-based differential expression analysis. RESULTS: We outline a statistical method for protein differential expression, based on a simple Binomial likelihood. By modeling peak intensities as binary, in terms of \\'presence/absence,\\' we enable the selection of proteins not typically amenable to quantitative analysis; e.g. \\'one-state\\' proteins that are present in one condition but absent in another. In addition, we present an analysis protocol that combines quantitative and presence/absence analysis of a given dataset in a principled way, resulting in a single list of selected proteins with a single-associated false discovery rate. AVAILABILITY: All R code available here: http://www.stat.tamu.edu/~adabney/share/xuan_code.zip.

Integrating atomistic molecular dynamics simulations, experiments and network analysis to study protein dynamics: strength in unity

Directory of Open Access Journals (Sweden)

Elena ePapaleo

2015-05-01

Full Text Available In the last years, we have been observing remarkable improvements in the field of protein dynamics. Indeed, we can now study protein dynamics in atomistic details over several timescales with a rich portfolio of experimental and computational techniques. On one side, this provides us with the possibility to validate simulation methods and physical models against a broad range of experimental observables. On the other side, it also allows a complementary and comprehensive view on protein structure and dynamics. What is needed now is a better understanding of the link between the dynamic properties that we observe and the functional properties of these important cellular machines. To make progresses in this direction, we need to improve the physical models used to describe proteins and solvent in molecular dynamics, as well as to strengthen the integration of experiments and simulations to overcome their own limitations. Moreover, now that we have the means to study protein dynamics in great details, we need new tools to understand the information embedded in the protein ensembles and in their dynamic signature. With this aim in mind, we should enrich the current tools for analysis of biomolecular simulations with attention to the effects that can be propagated over long distances and are often associated to important biological functions. In this context, approaches inspired by network analysis can make an important contribution to the analysis of molecular dynamics simulations.

Hydrogen Tunneling Links Protein Dynamics to Enzyme Catalysis

Science.gov (United States)

Klinman, Judith P.; Kohen, Amnon

2014-01-01

The relationship between protein dynamics and function is a subject of considerable contemporary interest. Although protein motions are frequently observed during ligand binding and release steps, the contribution of protein motions to the catalysis of bond making/breaking processes is more difficult to probe and verify. Here, we show how the quantum mechanical hydrogen tunneling associated with enzymatic C–H bond cleavage provides a unique window into the necessity of protein dynamics for achieving optimal catalysis. Experimental findings support a hierarchy of thermodynamically equilibrated motions that control the H-donor and -acceptor distance and active-site electrostatics, creating an ensemble of conformations suitable for H-tunneling. A possible extension of this view to methyl transfer and other catalyzed reactions is also presented. The impact of understanding these dynamics on the conceptual framework for enzyme activity, inhibitor/drug design, and biomimetic catalyst design is likely to be substantial. PMID:23746260

ISL1 protein transduction promotes cardiomyocyte differentiation from human embryonic stem cells.

Directory of Open Access Journals (Sweden)

Hananeh Fonoudi

Full Text Available BACKGROUND: Human embryonic stem cells (hESCs have the potential to provide an unlimited source of cardiomyocytes, which are invaluable resources for drug or toxicology screening, medical research, and cell therapy. Currently a number of obstacles exist such as the insufficient efficiency of differentiation protocols, which should be overcome before hESC-derived cardiomyocytes can be used for clinical applications. Although the differentiation efficiency can be improved by the genetic manipulation of hESCs to over-express cardiac-specific transcription factors, these differentiated cells are not safe enough to be applied in cell therapy. Protein transduction has been demonstrated as an alternative approach for increasing the efficiency of hESCs differentiation toward cardiomyocytes. METHODS: We present an efficient protocol for the differentiation of hESCs in suspension by direct introduction of a LIM homeodomain transcription factor, Islet1 (ISL1 recombinant protein into the cells. RESULTS: We found that the highest beating clusters were derived by continuous treatment of hESCs with 40 µg/ml recombinant ISL1 protein during days 1-8 after the initiation of differentiation. The treatment resulted in up to a 3-fold increase in the number of beating areas. In addition, the number of cells that expressed cardiac specific markers (cTnT, CONNEXIN 43, ACTININ, and GATA4 doubled. This protocol was also reproducible for another hESC line. CONCLUSIONS: This study has presented a new, efficient, and reproducible procedure for cardiomyocytes differentiation. Our results will pave the way for scaled up and controlled differentiation of hESCs to be used for biomedical applications in a bioreactor culture system.

Dynamics of proteins aggregation. II. Dynamic scaling in confined media

Science.gov (United States)

Zheng, Size; Shing, Katherine S.; Sahimi, Muhammad

2018-03-01

In this paper, the second in a series devoted to molecular modeling of protein aggregation, a mesoscale model of proteins together with extensive discontinuous molecular dynamics simulation is used to study the phenomenon in a confined medium. The medium, as a model of a crowded cellular environment, is represented by a spherical cavity, as well as cylindrical tubes with two aspect ratios. The aggregation process leads to the formation of β sheets and eventually fibrils, whose deposition on biological tissues is believed to be a major factor contributing to many neuro-degenerative diseases, such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis diseases. Several important properties of the aggregation process, including dynamic evolution of the total number of the aggregates, the mean aggregate size, and the number of peptides that contribute to the formation of the β sheets, have been computed. We show, similar to the unconfined media studied in Paper I [S. Zheng et al., J. Chem. Phys. 145, 134306 (2016)], that the computed properties follow dynamic scaling, characterized by power laws. The existence of such dynamic scaling in unconfined media was recently confirmed by experiments. The exponents that characterize the power-law dependence on time of the properties of the aggregation process in spherical cavities are shown to agree with those in unbounded fluids at the same protein density, while the exponents for aggregation in the cylindrical tubes exhibit sensitivity to the geometry of the system. The effects of the number of amino acids in the protein, as well as the size of the confined media, have also been studied. Similarities and differences between aggregation in confined and unconfined media are described, including the possibility of no fibril formation, if confinement is severe.

Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

International Nuclear Information System (INIS)

Ostlund, Cecilia; Guan, Tinglu; Figlewicz, Denise A.; Hays, Arthur P.; Worman, Howard J.; Gerace, Larry; Schirmer, Eric C.

2009-01-01

Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.

Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

Energy Technology Data Exchange (ETDEWEB)

Ostlund, Cecilia [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Guan, Tinglu [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Figlewicz, Denise A. [Department of Neurology, University of Michigan, Ann Arbor, MI 48109 (United States); Hays, Arthur P. [Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Worman, Howard J. [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Gerace, Larry [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Schirmer, Eric C., E-mail: e.schirmer@ed.ac.uk [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR (United Kingdom)

2009-11-13

Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.

A scalable double-barcode sequencing platform for characterization of dynamic protein-protein interactions.

Science.gov (United States)

Schlecht, Ulrich; Liu, Zhimin; Blundell, Jamie R; St Onge, Robert P; Levy, Sasha F

2017-05-25

Several large-scale efforts have systematically catalogued protein-protein interactions (PPIs) of a cell in a single environment. However, little is known about how the protein interactome changes across environmental perturbations. Current technologies, which assay one PPI at a time, are too low throughput to make it practical to study protein interactome dynamics. Here, we develop a highly parallel protein-protein interaction sequencing (PPiSeq) platform that uses a novel double barcoding system in conjunction with the dihydrofolate reductase protein-fragment complementation assay in Saccharomyces cerevisiae. PPiSeq detects PPIs at a rate that is on par with current assays and, in contrast with current methods, quantitatively scores PPIs with enough accuracy and sensitivity to detect changes across environments. Both PPI scoring and the bulk of strain construction can be performed with cell pools, making the assay scalable and easily reproduced across environments. PPiSeq is therefore a powerful new tool for large-scale investigations of dynamic PPIs.

Delayed nonlinear cournot and bertrand dynamics with product differentiation.

Science.gov (United States)

Matsumoto, Akio; Szidarovszky, Ferenc

2007-07-01

Dynamic duopolies will be examined with product differentiation and isoelastic price functions. We will first prove that under realistic conditions the equilibrium is always locally asymptotically stable. The stability can however be lost if the firms use delayed information in forming their best responses. Stability conditions are derived in special cases, and simulation results illustrate the complexity of the dynamism of the systems. Both price and quantity adjusting models are discussed.

Alteration of protein levels during influenza virus H1N1 infection in host cells: a proteomic survey of host and virus reveals differential dynamics.

Directory of Open Access Journals (Sweden)

Susann Kummer

Full Text Available We studied the dynamics of the proteome of influenza virus A/PR/8/34 (H1N1 infected Madin-Darby canine kidney cells up to 12 hours post infection by mass spectrometry based quantitative proteomics using the approach of stable isotope labeling by amino acids in cell culture (SILAC. We identified 1311 cell proteins and, apart from the proton channel M2, all major virus proteins. Based on their abundance two groups of virus proteins could be distinguished being in line with the function of the proteins in genesis and formation of new virions. Further, the data indicate a correlation between the amount of proteins synthesized and their previously determined copy number inside the viral particle. We employed bioinformatic approaches such as functional clustering, gene ontology, and pathway (KEGG enrichment tests to uncover co-regulated cellular protein sets, assigned the individual subsets to their biological function, and determined their interrelation within the progression of viral infection. For the first time we are able to describe dynamic changes of the cellular and, of note, the viral proteome in a time dependent manner simultaneously. Through cluster analysis, time dependent patterns of protein abundances revealed highly dynamic up- and/or down-regulation processes. Taken together our study provides strong evidence that virus infection has a major impact on the cell status at the protein level.

Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle*

Science.gov (United States)

Roy Choudhury, Swarup; Westfall, Corey S.; Laborde, John P.; Bisht, Naveen C.; Jez, Joseph M.; Pandey, Sona

2012-01-01

Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1–4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1–4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks. PMID:22474294

Protein Loop Dynamics Are Complex and Depend on the Motions of the Whole Protein

Directory of Open Access Journals (Sweden)

Michael T. Zimmermann

2012-04-01

Full Text Available We investigate the relationship between the motions of the same peptide loop segment incorporated within a protein structure and motions of free or end-constrained peptides. As a reference point we also compare against alanine chains having the same length as the loop. Both the analysis of atomic molecular dynamics trajectories and structure-based elastic network models, reveal no general dependence on loop length or on the number of solvent exposed residues. Rather, the whole structure affects the motions in complex ways that depend strongly and specifically on the tertiary structure of the whole protein. Both the Elastic Network Models and Molecular Dynamics confirm the differences in loop dynamics between the free and structured contexts; there is strong agreement between the behaviors observed from molecular dynamics and the elastic network models. There is no apparent simple relationship between loop mobility and its size, exposure, or position within a loop. Free peptides do not behave the same as the loops in the proteins. Surface loops do not behave as if they were random coils, and the tertiary structure has a critical influence upon the apparent motions. This strongly implies that entropy evaluation of protein loops requires knowledge of the motions of the entire protein structure.

On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

International Nuclear Information System (INIS)

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Fernandez-Alberti, Sebastian; Roitberg, Adrian E.

2015-01-01

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data

On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

Energy Technology Data Exchange (ETDEWEB)

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Fernandez-Alberti, Sebastian, E-mail: sfalberti@gmail.com [Universidad Nacional de Quilmes, Roque Saenz Peña 352, B1876BXD Bernal (Argentina); Roitberg, Adrian E. [Departments of Physics and Chemistry, University of Florida, Gainesville, Florida 32611 (United States)

2015-06-28

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.

Dynamic properties of motor proteins with two subunits

International Nuclear Information System (INIS)

Kolomeisky, Anatoly B; III, Hubert Phillips

2005-01-01

The dynamics of motor protein molecules consisting of two subunits is investigated using simple discrete stochastic models. Exact steady-state analytical expressions are obtained for velocities and dispersions for any number of intermediate states and conformations between the corresponding binding states of proteins. These models enable us to provide a detailed description and comparison of two different mechanisms of the motion of motor proteins along the linear tracks: the hand-over-hand mechanism, when the motion of subunits alternate; and the inchworm mechanism, when one subunit is always trailing another one. It is shown that the proteins in the hand-over-hand mechanism move faster and fluctuate more than the molecules in the inchworm mechanism. The effect of external forces on dynamic properties of motor proteins is also discussed. Finally, a quantitative method, based on experimental observations for single motor proteins, is proposed for distinguishing between two mechanisms of motion

Inhibition of Protein Farnesylation Arrests Adipogenesis and Affects PPARγ Expression and Activation in Differentiating Mesenchymal Stem Cells

Science.gov (United States)

Rivas, Daniel; Akter, Rahima; Duque, Gustavo

2007-01-01

Protein farnesylation is required for the activation of multiple proteins involved in cell differentiation and function. In white adipose tissue protein, farnesylation has shown to be essential for the successful differentiation of preadipocytes into adipocytes. We hypothesize that protein farnesylation is required for PPARγ2 expression and activation, and therefore for the differentiation of human mesenchymal stem cells (MSCs) into adipocytes. MSCs were plated and induced to differentiate into adipocytes for three weeks. Differentiating cells were treated with either an inhibitor of farnesylation (FTI-277) or vehicle alone. The effect of inhibition of farnesylation in differentiating adipocytes was determined by oil red O staining. Cell survival was quantified using MTS Formazan. Additionally, nuclear extracts were obtained and prelamin A, chaperon protein HDJ-2, PPARγ, and SREBP-1 were determined by western blot. Finally, DNA binding PPARγ activity was determined using an ELISA-based PPARγ activation quantification method. Treatment with an inhibitor of farnesylation (FTI-277) arrests adipogenesis without affecting cell survival. This effect was concomitant with lower levels of PPARγ expression and activity. Finally, accumulation of prelamin A induced an increased proportion of mature SREBP-1 which is known to affect PPARγ activity. In summary, inhibition of protein farnesylation arrests the adipogenic differentiation of MSCs and affects PPARγ expression and activity. PMID:18274630

Differentially expressed proteins among normal cervix, cervical intraepithelial neoplasia and cervical squamous cell carcinoma.

Science.gov (United States)

Zhao, Q; He, Y; Wang, X-L; Zhang, Y-X; Wu, Y-M

2015-08-01

To explore the differentially expressed proteins in normal cervix, cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC) tissues by differential proteomics technique. Cervical tissues (including normal cervix, CIN and CSCC) were collected in Department of Gynecologic Oncology of Beijing Obstetrics and Gynecology Hospital. Two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) and DeCyder software were used to detect the differentially expressed proteins. Matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) was used to identify the differentially expressed proteins. Western blot (WB) and immunohistochemistry (IHC) were performed to validate the expressions of selected proteins among normal cervix, CIN and CSCC. 2-D DIGE images with high resolution and good repeatability were obtained. Forty-six differentially expressed proteins (27 up-regulated and 19 down-regulated) were differentially expressed among the normal cervix, CIN and CSCC. 26 proteins were successfully identified by MALDI-TOF/TOF MS. S100A9 (S100 calcium-binding protein A9) was the most significantly up-regulated protein. Eukaryotic elongation factor 1-alpha-1 (eEF1A1) was the most significantly down-regulated protein. Pyruvate kinase isozymes M2 (PKM2) was both up-regulated and down-regulated. The results of WB showed that with the increase in the severity of cervical lesions, the expression of S100A9 protein was significantly increased among the three groups (P = 0.010). The expression of eEF1A1 was reduced but without significant difference (P = 0.861). The expression of PKM2 was significantly reduced (P = 0.000). IHC showed that protein S100A9 was mainly expressed in the cytoplasm, and its positive expression rate was 20.0 % in normal cervix, 70.0 % in CIN and 100.0 % in CSCC, with a significant difference among them (P = 0.006). eEF1A1 was mainly expressed in the cell plasma, and its

Discriminating lysosomal membrane protein types using dynamic neural network.

Science.gov (United States)

Tripathi, Vijay; Gupta, Dwijendra Kumar

2014-01-01

This work presents a dynamic artificial neural network methodology, which classifies the proteins into their classes from their sequences alone: the lysosomal membrane protein classes and the various other membranes protein classes. In this paper, neural networks-based lysosomal-associated membrane protein type prediction system is proposed. Different protein sequence representations are fused to extract the features of a protein sequence, which includes seven feature sets; amino acid (AA) composition, sequence length, hydrophobic group, electronic group, sum of hydrophobicity, R-group, and dipeptide composition. To reduce the dimensionality of the large feature vector, we applied the principal component analysis. The probabilistic neural network, generalized regression neural network, and Elman regression neural network (RNN) are used as classifiers and compared with layer recurrent network (LRN), a dynamic network. The dynamic networks have memory, i.e. its output depends not only on the input but the previous outputs also. Thus, the accuracy of LRN classifier among all other artificial neural networks comes out to be the highest. The overall accuracy of jackknife cross-validation is 93.2% for the data-set. These predicted results suggest that the method can be effectively applied to discriminate lysosomal associated membrane proteins from other membrane proteins (Type-I, Outer membrane proteins, GPI-Anchored) and Globular proteins, and it also indicates that the protein sequence representation can better reflect the core feature of membrane proteins than the classical AA composition.

Serum immune-related proteins are differentially expressed during hibernation in the American black bear.

Directory of Open Access Journals (Sweden)

Brian A Chow

Full Text Available Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α2-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears.

Bioluminescence resonance energy transfer system for measuring dynamic protein-protein interactions in bacteria.

Science.gov (United States)

Cui, Boyu; Wang, Yao; Song, Yunhong; Wang, Tietao; Li, Changfu; Wei, Yahong; Luo, Zhao-Qing; Shen, Xihui

2014-05-20

Protein-protein interactions are important for virtually every biological process, and a number of elegant approaches have been designed to detect and evaluate such interactions. However, few of these methods allow the detection of dynamic and real-time protein-protein interactions in bacteria. Here we describe a bioluminescence resonance energy transfer (BRET) system based on the bacterial luciferase LuxAB. We found that enhanced yellow fluorescent protein (eYFP) accepts the emission from LuxAB and emits yellow fluorescence. Importantly, BRET occurred when LuxAB and eYFP were fused, respectively, to the interacting protein pair FlgM and FliA. Furthermore, we observed sirolimus (i.e., rapamycin)-inducible interactions between FRB and FKBP12 and a dose-dependent abolishment of such interactions by FK506, the ligand of FKBP12. Using this system, we showed that osmotic stress or low pH efficiently induced multimerization of the regulatory protein OmpR and that the multimerization induced by low pH can be reversed by a neutralizing agent, further indicating the usefulness of this system in the measurement of dynamic interactions. This method can be adapted to analyze dynamic protein-protein interactions and the importance of such interactions in bacterial processes such as development and pathogenicity. Real-time measurement of protein-protein interactions in prokaryotes is highly desirable for determining the roles of protein complex in the development or virulence of bacteria, but methods that allow such measurement are not available. Here we describe the development of a bioluminescence resonance energy transfer (BRET) technology that meets this need. The use of endogenous excitation light in this strategy circumvents the requirement for the sophisticated instrument demanded by standard fluorescence resonance energy transfer (FRET). Furthermore, because the LuxAB substrate decanal is membrane permeable, the assay can be performed without lysing the bacterial cells

Effect of Wnt-1 inducible signaling pathway protein-2 (WISP-2/CCN5), a downstream protein of Wnt signaling, on adipocyte differentiation

International Nuclear Information System (INIS)

Inadera, Hidekuni; Shimomura, Akiko; Tachibana, Shinjiro

2009-01-01

Wnt signaling negatively regulates adipocyte differentiation, and ectopic expression of Wnt-1 in 3T3-L1 cells induces several downstream molecules of Wnt signaling, including Wnt-1 inducible signaling pathway protein (WISP)-2. In this study, we examined the role of WISP-2 in the process of adipocyte differentiation using an in vitro cell culture system. In the differentiation of 3T3-L1 cells, WISP-2 expression was observed in growing cells and declined thereafter. In the mitotic clonal expansion phase of adipocyte differentiation, WISP-2 expression was transiently down-regulated concurrently with up-regulation of CCAAT/enhancer-binding protein δ expression. Treatment of 3T3-L1 cells in the differentiation medium with lithium, an activator of Wnt signaling, inhibited the differentiation process with concomitant induction of WISP-2. Treatment of differentiated cells with lithium induced de-differentiation as evidenced by profound reduction of peroxisome proliferator-activator receptor γ expression and concomitant induction of WISP-2. However, de-differentiation of differentiated cells induced by tumor necrosis factor-α did not induce WISP-2 expression. To directly examine the effect of WISP-2 on adipocyte differentiation, 3T3-L1 cells were infected with a retrovirus carrying WISP-2. Although forced expression of WISP-2 inhibited preadipocyte proliferation, it had no effect on adipocyte differentiation. Thus, although WISP-2 is a downstream protein of Wnt signaling, the role of WISP-2 on adipocyte differentiation may be marginal, at least in this in vitro culture model.

Time-resolved infrared studies of protein conformational dynamics

Energy Technology Data Exchange (ETDEWEB)

Woodruff, W.H.; Causgrove, T.P.; Dyer, R.B. [Los Alamos National Laboratory, NM (United States); Callender, R.H. [Univ. of New York, NY (United States)

1994-12-01

We have demonstrated that TRIR in the amide I region gives structural information regarding protein conformational changes in realtime, both on processes involved in the development of the functional structure (protein folding) and on protein structural changes that accompany the functional dynamics of the native structure. Assignment of many of the amide I peaks to specific amide or sidechain structures will require much additional effort. Specifically, the congestion and complexity of the protein vibrational spectra dictate that isotope studies are an absolute requirement for more than a qualitative notion of the structural interpretation of these measurements. It is clear, however, that enormous potential exists for elucidating structural relaxation dynamics and energetics with a high degree of structural specificity using this approach.

Molecular dynamics of surfactant protein C

DEFF Research Database (Denmark)

Ramírez, Eunice; Santana, Alberto; Cruz, Anthony

2006-01-01

Surfactant protein C (SP-C) is a membrane-associated protein essential for normal respiration. It has been found that the alpha-helix form of SP-C can undergo, under certain conditions, a transformation from an alpha-helix to a beta-strand conformation that closely resembles amyloid fibrils, which...... are possible contributors to the pathogenesis of pulmonary alveolar proteinosis. Molecular dynamics simulations using the NAMD2 package were performed for systems containing from one to seven SP-C molecules to study their behavior in water. The results of our simulations show that unfolding of the protein...

Dynamic Pricing Competition with Strategic Customers Under Vertical Product Differentiation

OpenAIRE

Qian Liu; Dan Zhang

2013-01-01

We consider dynamic pricing competition between two firms offering vertically differentiated products to strategic customers who are intertemporal utility maximizers. We show that price skimming arises as the unique pure-strategy Markov perfect equilibrium in the game under a simple condition. Our results highlight the asymmetric effect of strategic customer behavior on quality-differentiated firms. Even though the profit of either firm decreases as customers become more strategic, the low-qu...

Quantitative proteome and phosphoproteome analyses of Streptomyces coelicolor reveal proteins and phosphoproteins modulating differentiation and secondary metabolism

DEFF Research Database (Denmark)

Rioseras, Beatriz; Sliaha, Pavel V; Gorshkov, Vladimir

2018-01-01

identified and quantified 3461 proteins corresponding to 44.3% of the S. coelicolor proteome across three developmental stages: vegetative hypha (MI); secondary metabolite producing hyphae (MII); and sporulating hyphae. A total of 1350 proteins exhibited more than 2-fold expression changes during....../Thr/Tyr kinases, making this genus an outstanding model for the study of bacterial protein phosphorylation events. We used mass spectrometry based quantitative proteomics and phosphoproteomics to characterize bacterial differentiation and activation of secondary metabolism of Streptomyces coelicolor. We...... the bacterial differentiation process. These proteins include 136 regulators (transcriptional regulators, transducers, Ser/Thr/Tyr kinases, signalling proteins), as well as 542 putative proteins with no clear homology to known proteins which are likely to play a role in differentiation and secondary metabolism...

Hydration and temperature interdependence of protein picosecond dynamics.

Science.gov (United States)

Lipps, Ferdinand; Levy, Seth; Markelz, A G

2012-05-14

We investigate the nature of the solvent motions giving rise to the rapid temperature dependence of protein picoseconds motions at 220 K, often referred to as the protein dynamical transition. The interdependence of picoseconds dynamics on hydration and temperature is examined using terahertz time domain spectroscopy to measure the complex permittivity in the 0.2-2.0 THz range for myoglobin. Both the real and imaginary parts of the permittivity over the frequency range measured have a strong temperature dependence at >0.27 h (g water per g protein), however the permittivity change is strongest for frequencies 1 THz, and 0.27 h for frequencies <1 THz. The data are consistent with the dynamical transition solvent fluctuations requiring only clusters of ~5 water molecules, whereas the enhancement of lowest frequency motions requires a fully spanning water network. This journal is © the Owner Societies 2012

Differential expression of proteins and phosphoproteins during larval metamorphosis of the polychaete Capitella sp. I

Directory of Open Access Journals (Sweden)

Qian Pei-Yuan

2011-09-01

Full Text Available Abstract Background The spontaneous metamorphosis of the polychaete Capitella sp. I larvae into juveniles requires minor morphological changes, including segment formation, body elongation, and loss of cilia. In this study, we investigated changes in the expression patterns of both proteins and phosphoproteins during the transition from larvae to juveniles in this species. We used two-dimensional gel electrophoresis (2-DE followed by multiplex fluorescent staining and MALDI-TOF mass spectrometry analysis to identify the differentially expressed proteins as well as the protein and phosphoprotein profiles of both competent larvae and juveniles. Results Twenty-three differentially expressed proteins were identified in the two developmental stages. Expression patterns of two of those proteins were examined at the protein level by Western blot analysis while seven were further studied at the mRNA level by real-time PCR. Results showed that proteins related to cell division, cell migration, energy storage and oxidative stress were plentifully expressed in the competent larvae; in contrast, proteins involved in oxidative metabolism and transcriptional regulation were abundantly expressed in the juveniles. Conclusion It is likely that these differentially expressed proteins are involved in regulating the larval metamorphosis process and can be used as protein markers for studying molecular mechanisms associated with larval metamorphosis in polychaetes.

A VESICLE TRAFFICKING PROTEIN αSNAP REGULATES PANETH CELL DIFFERENTIATION IN VIVO

Science.gov (United States)

Lechuga, Susana; Naydenov, Nayden G.; Feygin, Alex; Jimenez, Antonio J.; Ivanov, Andrei I.

2017-01-01

A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo. PMID:28359759

Organization and Dynamics of Receptor Proteins in a Plasma Membrane.

Science.gov (United States)

Koldsø, Heidi; Sansom, Mark S P

2015-11-25

The interactions of membrane proteins are influenced by their lipid environment, with key lipid species able to regulate membrane protein function. Advances in high-resolution microscopy can reveal the organization and dynamics of proteins and lipids within living cells at resolutions membranes of in vivo-like complexity. We explore the dynamics of proteins and lipids in crowded and complex plasma membrane models, thereby closing the gap in length and complexity between computations and experiments. Our simulations provide insights into the mutual interplay between lipids and proteins in determining mesoscale (20-100 nm) fluctuations of the bilayer, and in enabling oligomerization and clustering of membrane proteins.

Dynamics of Hippocampal Protein Expression During Long-term Spatial Memory Formation*

Science.gov (United States)

Borovok, Natalia; Nesher, Elimelech; Levin, Yishai; Reichenstein, Michal; Pinhasov, Albert

2016-01-01

Spatial memory depends on the hippocampus, which is particularly vulnerable to aging. This vulnerability has implications for the impairment of navigation capacities in older people, who may show a marked drop in performance of spatial tasks with advancing age. Contemporary understanding of long-term memory formation relies on molecular mechanisms underlying long-term synaptic plasticity. With memory acquisition, activity-dependent changes occurring in synapses initiate multiple signal transduction pathways enhancing protein turnover. This enhancement facilitates de novo synthesis of plasticity related proteins, crucial factors for establishing persistent long-term synaptic plasticity and forming memory engrams. Extensive studies have been performed to elucidate molecular mechanisms of memory traces formation; however, the identity of plasticity related proteins is still evasive. In this study, we investigated protein turnover in mouse hippocampus during long-term spatial memory formation using the reference memory version of radial arm maze (RAM) paradigm. We identified 1592 proteins, which exhibited a complex picture of expression changes during spatial memory formation. Variable linear decomposition reduced significantly data dimensionality and enriched three principal factors responsible for variance of memory-related protein levels at (1) the initial phase of memory acquisition (165 proteins), (2) during the steep learning improvement (148 proteins), and (3) the final phase of the learning curve (123 proteins). Gene ontology and signaling pathways analysis revealed a clear correlation between memory improvement and learning phase-curbed expression profiles of proteins belonging to specific functional categories. We found differential enrichment of (1) neurotrophic factors signaling pathways, proteins regulating synaptic transmission, and actin microfilament during the first day of the learning curve; (2) transcription and translation machinery, protein

Dynamics of Hippocampal Protein Expression During Long-term Spatial Memory Formation.

Science.gov (United States)

Borovok, Natalia; Nesher, Elimelech; Levin, Yishai; Reichenstein, Michal; Pinhasov, Albert; Michaelevski, Izhak

2016-02-01

Spatial memory depends on the hippocampus, which is particularly vulnerable to aging. This vulnerability has implications for the impairment of navigation capacities in older people, who may show a marked drop in performance of spatial tasks with advancing age. Contemporary understanding of long-term memory formation relies on molecular mechanisms underlying long-term synaptic plasticity. With memory acquisition, activity-dependent changes occurring in synapses initiate multiple signal transduction pathways enhancing protein turnover. This enhancement facilitates de novo synthesis of plasticity related proteins, crucial factors for establishing persistent long-term synaptic plasticity and forming memory engrams. Extensive studies have been performed to elucidate molecular mechanisms of memory traces formation; however, the identity of plasticity related proteins is still evasive. In this study, we investigated protein turnover in mouse hippocampus during long-term spatial memory formation using the reference memory version of radial arm maze (RAM) paradigm. We identified 1592 proteins, which exhibited a complex picture of expression changes during spatial memory formation. Variable linear decomposition reduced significantly data dimensionality and enriched three principal factors responsible for variance of memory-related protein levels at (1) the initial phase of memory acquisition (165 proteins), (2) during the steep learning improvement (148 proteins), and (3) the final phase of the learning curve (123 proteins). Gene ontology and signaling pathways analysis revealed a clear correlation between memory improvement and learning phase-curbed expression profiles of proteins belonging to specific functional categories. We found differential enrichment of (1) neurotrophic factors signaling pathways, proteins regulating synaptic transmission, and actin microfilament during the first day of the learning curve; (2) transcription and translation machinery, protein

Discovery of protein profiles for differentiated thyroid cancer using SELDI TOF MS

International Nuclear Information System (INIS)

Yoon, Joon Kee; Lee, Myung Hoon; Joh, Chul Woo; Yoon, Seok Nam; Soh, Eui Young

2003-01-01

Low sensitivity of diagnostic whole body iodine scintigraphy and intermediate range of serum thyroglobulin (Tg) with or without anti-Tg antibody make it difficult to select the patients with differentiated thyroid cancer who need further treatment. Surfaced Enhanced Laser Desorption /Ionization - Time of Flight - Mass Spectrometry (SELDI TOF MS) is a useful method to evaluate cancer proteome, biomarkers and patterns of biomarkers. In this preliminary study, we evaluated and developed protein profiles for the discrimination between patients with differentiated thyroid cancer and non-cancer controls using SELDI technology. Serum samples from 10 healthy controls and from 14 patients with papillary thyroid cancer before thyroidectomy were analyzed by SELDI MS. Multiple protein peaks detected were analyzed by the computer software to develop a classifier for separating cancer patients form controls. The classifier was then challenged to 24 serum samples to determine the validity and accuracy of the classification system. All patients with papillary thyroid cancer had no other concomitant cancer or thyroiditis. Their serum Tg concentration was 55.8 (1.5 - 249.7) and 2 patients had extra-thyroidal extension. According to the SELDI analysis, protein peaks at 3696 Da, 4178 Da, and 8149 Da were more prominent in cancer patients than controls in various degrees. Among those, protein peak at 4178 Da was determined as classifier by computer software, and the sensitivity, specificity and accuracy for discrimination of cancer patients from controls was 92.9% (13/14), 90% (9/10) and 91.7% respectively. This preliminary study suggests that serum protein profiles of differentiated thyroid cancer can be used for differentiation between cancer patients and non-cancer controls. And further clinical studies in various test sets will offer useful information in selecting patients who require treatment

Discovery of protein profiles for differentiated thyroid cancer using SELDI TOF MS

Energy Technology Data Exchange (ETDEWEB)

Yoon, Joon Kee; Lee, Myung Hoon; Joh, Chul Woo; Yoon, Seok Nam; Soh, Eui Young [College of Medicine, Univ. of Ajou, Suwon (Korea, Republic of)

2003-07-01

Low sensitivity of diagnostic whole body iodine scintigraphy and intermediate range of serum thyroglobulin (Tg) with or without anti-Tg antibody make it difficult to select the patients with differentiated thyroid cancer who need further treatment. Surfaced Enhanced Laser Desorption /Ionization - Time of Flight - Mass Spectrometry (SELDI TOF MS) is a useful method to evaluate cancer proteome, biomarkers and patterns of biomarkers. In this preliminary study, we evaluated and developed protein profiles for the discrimination between patients with differentiated thyroid cancer and non-cancer controls using SELDI technology. Serum samples from 10 healthy controls and from 14 patients with papillary thyroid cancer before thyroidectomy were analyzed by SELDI MS. Multiple protein peaks detected were analyzed by the computer software to develop a classifier for separating cancer patients form controls. The classifier was then challenged to 24 serum samples to determine the validity and accuracy of the classification system. All patients with papillary thyroid cancer had no other concomitant cancer or thyroiditis. Their serum Tg concentration was 55.8 (1.5 - 249.7) and 2 patients had extra-thyroidal extension. According to the SELDI analysis, protein peaks at 3696 Da, 4178 Da, and 8149 Da were more prominent in cancer patients than controls in various degrees. Among those, protein peak at 4178 Da was determined as classifier by computer software, and the sensitivity, specificity and accuracy for discrimination of cancer patients from controls was 92.9% (13/14), 90% (9/10) and 91.7% respectively. This preliminary study suggests that serum protein profiles of differentiated thyroid cancer can be used for differentiation between cancer patients and non-cancer controls. And further clinical studies in various test sets will offer useful information in selecting patients who require treatment.

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

Directory of Open Access Journals (Sweden)

Markus M. Knodel

2018-01-01

Full Text Available Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface.

Science.gov (United States)

Knodel, Markus M; Nägel, Arne; Reiter, Sebastian; Vogel, Andreas; Targett-Adams, Paul; McLauchlan, John; Herrmann, Eva; Wittum, Gabriel

2018-01-08

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

Science.gov (United States)

Nägel, Arne; Reiter, Sebastian; Vogel, Andreas; McLauchlan, John; Herrmann, Eva; Wittum, Gabriel

2018-01-01

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles. PMID:29316722

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

KAUST Repository

Knodel, Markus

2018-01-08

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

Identification of differentially expressed proteins during human urinary bladder cancer progression.

Science.gov (United States)

Memon, Ashfaque A; Chang, Jong W; Oh, Bong R; Yoo, Yung J

2005-01-01

Comparative proteome analysis was performed between RT4 (grade-1) and T24 (grade-3) bladder cancer cell lines, in an attempt to identify differentially expressed proteins during bladder cancer progression. Among those relatively abundant proteins, seven spots changed more than two-fold reproducibly and identified by peptide mass fingerprinting using mass spectrometry and database search. We found most extensive and reproducible down-regulation of NADP dependent isocitrate dehydrogenase cytoplasmic (IDPc) and peroxiredoxin-II (Prx-II), in poorly differentiated T24 compared to well-differentiated RT4 bladder cancer cell line. Subsequent Western blotting analysis of human biopsy samples from bladder cancer patient revealed significant loss of IDPc and Prx-II in more advance tumor samples, in agreement with data on cell lines. These results suggest that loss of IDPc and Prx-II during tumor development may involve in tumor progression and metastasis. However, additional investigations are needed on large number of human samples to further verify these findings.

How proteins modify water dynamics

Science.gov (United States)

Persson, Filip; Söderhjelm, Pär; Halle, Bertil

2018-06-01

Much of biology happens at the protein-water interface, so all dynamical processes in this region are of fundamental importance. Local structural fluctuations in the hydration layer can be probed by 17O magnetic relaxation dispersion (MRD), which, at high frequencies, measures the integral of a biaxial rotational time correlation function (TCF)—the integral rotational correlation time. Numerous 17O MRD studies have demonstrated that this correlation time, when averaged over the first hydration shell, is longer than in bulk water by a factor 3-5. This rotational perturbation factor (RPF) has been corroborated by molecular dynamics simulations, which can also reveal the underlying molecular mechanisms. Here, we address several outstanding problems in this area by analyzing an extensive set of molecular dynamics data, including four globular proteins and three water models. The vexed issue of polarity versus topography as the primary determinant of hydration water dynamics is resolved by establishing a protein-invariant exponential dependence of the RPF on a simple confinement index. We conclude that the previously observed correlation of the RPF with surface polarity is a secondary effect of the correlation between polarity and confinement. Water rotation interpolates between a perturbed but bulk-like collective mechanism at low confinement and an exchange-mediated orientational randomization (EMOR) mechanism at high confinement. The EMOR process, which accounts for about half of the RPF, was not recognized in previous simulation studies, where only the early part of the TCF was examined. Based on the analysis of the experimentally relevant TCF over its full time course, we compare simulated and measured RPFs, finding a 30% discrepancy attributable to force field imperfections. We also compute the full 17O MRD profile, including the low-frequency dispersion produced by buried water molecules. Computing a local RPF for each hydration shell, we find that the

Differentiation of Vitis vinifera varieties by MALDI-MS analysis of the grape seed proteins.

Science.gov (United States)

Pesavento, Ivana Chiara; Bertazzo, Antonella; Flamini, Riccardo; Vedova, Antonio Dalla; De Rosso, Mirko; Seraglia, Roberta; Traldi, Pietro

2008-02-01

Until now the study of pathogenic related proteins in grape juice and wine, performed by ESI-MS, LC/ESI-MS, and MALDI/MS, has been proposed for differentiation of varieties. In fact, chitinases and thaumatin-like proteins persist through the vinification process and cause hazes and sediments in bottled wines. An additional instrument, potentially suitable for the grape varieties differentiation, has been developed by MALDI/MS for the grape seed protein analysis. The hydrosoluble protein profiles of seeds extract from three different Vitis vinifera grape (red and white) varieties were analyzed and compared. In order to evaluate the environmental conditions and harvest effects, the seed protein profiles of one grape variety from different locations and harvests were studied. (c) 2008 John Wiley & Sons, Ltd.

Evolutionary dynamics of protein domain architecture in plants

Directory of Open Access Journals (Sweden)

Zhang Xue-Cheng

2012-01-01

Full Text Available Abstract Background Protein domains are the structural, functional and evolutionary units of the protein. Protein domain architectures are the linear arrangements of domain(s in individual proteins. Although the evolutionary history of protein domain architecture has been extensively studied in microorganisms, the evolutionary dynamics of domain architecture in the plant kingdom remains largely undefined. To address this question, we analyzed the lineage-based protein domain architecture content in 14 completed green plant genomes. Results Our analyses show that all 14 plant genomes maintain similar distributions of species-specific, single-domain, and multi-domain architectures. Approximately 65% of plant domain architectures are universally present in all plant lineages, while the remaining architectures are lineage-specific. Clear examples are seen of both the loss and gain of specific protein architectures in higher plants. There has been a dynamic, lineage-wise expansion of domain architectures during plant evolution. The data suggest that this expansion can be largely explained by changes in nuclear ploidy resulting from rounds of whole genome duplications. Indeed, there has been a decrease in the number of unique domain architectures when the genomes were normalized into a presumed ancestral genome that has not undergone whole genome duplications. Conclusions Our data show the conservation of universal domain architectures in all available plant genomes, indicating the presence of an evolutionarily conserved, core set of protein components. However, the occurrence of lineage-specific domain architectures indicates that domain architecture diversity has been maintained beyond these core components in plant genomes. Although several features of genome-wide domain architecture content are conserved in plants, the data clearly demonstrate lineage-wise, progressive changes and expansions of individual protein domain architectures, reinforcing

Bidirectional lipid droplet velocities are controlled by differential binding strengths of HCV core DII protein.

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Rodney K Lyn

Full Text Available Host cell lipid droplets (LD are essential in the hepatitis C virus (HCV life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein's lipid binding domain II (DII-core induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV.

Integrating atomistic molecular dynamics simulations, experiments, and network analysis to study protein dynamics

DEFF Research Database (Denmark)

Papaleo, Elena

2015-01-01

that we observe and the functional properties of these important cellular machines. To make progresses in this direction, we need to improve the physical models used to describe proteins and solvent in molecular dynamics, as well as to strengthen the integration of experiments and simulations to overcome...... with the possibility to validate simulation methods and physical models against a broad range of experimental observables. On the other side, it also allows a complementary and comprehensive view on protein structure and dynamics. What is needed now is a better understanding of the link between the dynamic properties...... simulations with attention to the effects that can be propagated over long distances and are often associated to important biological functions. In this context, approaches inspired by network analysis can make an important contribution to the analysis of molecular dynamics simulations....

Dynamics in electron transfer protein complexes

OpenAIRE

Bashir, Qamar

2010-01-01

Recent studies have provided experimental evidence for the existence of an encounter complex, a transient intermediate in the formation of protein complexes. We have used paramagnetic relaxation enhancement NMR spectroscopy in combination with Monte Carlo simulations to characterize and visualize the ensemble of encounter orientations in the short-lived electron transfer complex of yeast Cc and CcP. The complete conformational space sampled by the protein molecules during the dynamic part of ...

Flexibility damps macromolecular crowding effects on protein folding dynamics: Application to the murine prion protein (121-231)

Science.gov (United States)

Bergasa-Caceres, Fernando; Rabitz, Herschel A.

2014-01-01

A model of protein folding kinetics is applied to study the combined effects of protein flexibility and macromolecular crowding on protein folding rate and stability. It is found that the increase in stability and folding rate promoted by macromolecular crowding is damped for proteins with highly flexible native structures. The model is applied to the folding dynamics of the murine prion protein (121-231). It is found that the high flexibility of the native isoform of the murine prion protein (121-231) reduces the effects of macromolecular crowding on its folding dynamics. The relevance of these findings for the pathogenic mechanism are discussed.

Expression of Iron-Related Proteins Differentiate Non-Cancerous and Cancerous Breast Tumors

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Sara Pizzamiglio

2017-02-01

Full Text Available We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA or reverse-phase protein array (RPPA, were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012. The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5, signal transducer and activator of transcription 3 (STAT3, bone morphogenetic protein 6 (BMP6, cluster of differentiation 74 (CD74, transferrin receptor (TFRC, inhibin alpha (INHA, and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88, CD74, iron exporter ferroportin (FPN, high mobility group box 1 (HMGB1, STAT3_pS727, TFRC, ferritin heavy chain (FTH, and ferritin light chain (FTL. Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer.

Proteins Differentially Expressed in the Pancreas of Hepatic Alcohol Dehydrogenase-Deficient Deer Mice Fed Ethanol For 3 Months.

Science.gov (United States)

Bhopale, Kamlesh K; Amer, Samir M; Kaphalia, Lata; Soman, Kizhake V; Wiktorowicz, John E; Shakeel Ansari, Ghulam A; Kaphalia, Bhupendra S

2017-07-01

The aim of this study was to identify differentially expressed proteins in the pancreatic tissue of hepatic alcohol dehydrogenase-deficient deer mice fed ethanol to understand metabolic basis and mechanism of alcoholic chronic pancreatitis. Mice were fed liquid diet containing 3.5 g% ethanol daily for 3 months, and differentially expressed pancreatic proteins were identified by protein separation using 2-dimensional gel electrophoresis and identification by mass spectrometry. Nineteen differentially expressed proteins were identified by applying criteria established for protein identification in proteomics. An increased abundance was found for ribosome-binding protein 1, 60S ribosomal protein L31-like isoform 1, histone 4, calcium, and adenosine triphosphate (ATP) binding proteins and the proteins involved in antiapoptotic processes and endoplasmic reticulum function, stress, and/or homeostasis. Low abundance was found for endoA cytokeratin, 40S ribosomal protein SA, amylase 2b isoform precursor, serum albumin, and ATP synthase subunit β and the proteins involved in cell motility, structure, and conformation. Chronic ethanol feeding in alcohol dehydrogenase-deficient deer mice differentially expresses pancreatic functional and structural proteins, which can be used to develop biomarker(s) of alcoholic chronic pancreatitis, particularly amylase 2b precursor, and 60 kDa heat shock protein and those involved in ATP synthesis and blood osmotic pressure.

Quantitative analysis of differentially expressed saliva proteins in human immunodeficiency virus type 1 (HIV-1) infected individuals

International Nuclear Information System (INIS)

Zhang, Nawei; Zhang, Zhenyu; Feng, Shan; Wang, Qingtao; Malamud, Daniel; Deng, Haiteng

2013-01-01

Highlights: ► A high-throughput method for profiling and quantification of the differentially expressed proteins in saliva samples was developed. ► Identified that DMBT1, S100A7, S100A8, S100A9 and alpha defensin were up-regulated in saliva from HIV-1 seropositive patients. ► Established analytical strategies are translatable to the clinical setting. -- Abstract: In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity

Quantitative analysis of differentially expressed saliva proteins in human immunodeficiency virus type 1 (HIV-1) infected individuals

Energy Technology Data Exchange (ETDEWEB)

Zhang, Nawei; Zhang, Zhenyu [Beijing Chaoyang Hospital Affiliated Capital Medical University, Beijing (China); Feng, Shan [MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing (China); Wang, Qingtao [Beijing Chaoyang Hospital Affiliated Capital Medical University, Beijing (China); Malamud, Daniel [NYU College of Dentistry, 345 East 24th Street, New York, NY 10010 (United States); Deng, Haiteng, E-mail: dht@mail.tsinghua.edu.cn [MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing (China)

2013-04-24

Highlights: ► A high-throughput method for profiling and quantification of the differentially expressed proteins in saliva samples was developed. ► Identified that DMBT1, S100A7, S100A8, S100A9 and alpha defensin were up-regulated in saliva from HIV-1 seropositive patients. ► Established analytical strategies are translatable to the clinical setting. -- Abstract: In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity.

Differential expression of proteins and phosphoproteins during larval metamorphosis of the polychaete Capitella sp. I

KAUST Repository

Chandramouli, Kondethimmanahalli

2011-09-03

Background: The spontaneous metamorphosis of the polychaete Capitella sp. I larvae into juveniles requires minor morphological changes, including segment formation, body elongation, and loss of cilia. In this study, we investigated changes in the expression patterns of both proteins and phosphoproteins during the transition from larvae to juveniles in this species. We used two-dimensional gel electrophoresis (2-DE) followed by multiplex fluorescent staining and MALDI-TOF mass spectrometry analysis to identify the differentially expressed proteins as well as the protein and phosphoprotein profiles of both competent larvae and juveniles.Results: Twenty-three differentially expressed proteins were identified in the two developmental stages. Expression patterns of two of those proteins were examined at the protein level by Western blot analysis while seven were further studied at the mRNA level by real-time PCR. Results showed that proteins related to cell division, cell migration, energy storage and oxidative stress were plentifully expressed in the competent larvae; in contrast, proteins involved in oxidative metabolism and transcriptional regulation were abundantly expressed in the juveniles.Conclusion: It is likely that these differentially expressed proteins are involved in regulating the larval metamorphosis process and can be used as protein markers for studying molecular mechanisms associated with larval metamorphosis in polychaetes. © 2011 Chandramouli et al; licensee BioMed Central Ltd.

Differential expression of proteins and phosphoproteins during larval metamorphosis of the polychaete Capitella sp. I

KAUST Repository

Chandramouli, Kondethimmanahalli; Soo, Lisa; Qian, Pei-Yuan

2011-01-01

Background: The spontaneous metamorphosis of the polychaete Capitella sp. I larvae into juveniles requires minor morphological changes, including segment formation, body elongation, and loss of cilia. In this study, we investigated changes in the expression patterns of both proteins and phosphoproteins during the transition from larvae to juveniles in this species. We used two-dimensional gel electrophoresis (2-DE) followed by multiplex fluorescent staining and MALDI-TOF mass spectrometry analysis to identify the differentially expressed proteins as well as the protein and phosphoprotein profiles of both competent larvae and juveniles.Results: Twenty-three differentially expressed proteins were identified in the two developmental stages. Expression patterns of two of those proteins were examined at the protein level by Western blot analysis while seven were further studied at the mRNA level by real-time PCR. Results showed that proteins related to cell division, cell migration, energy storage and oxidative stress were plentifully expressed in the competent larvae; in contrast, proteins involved in oxidative metabolism and transcriptional regulation were abundantly expressed in the juveniles.Conclusion: It is likely that these differentially expressed proteins are involved in regulating the larval metamorphosis process and can be used as protein markers for studying molecular mechanisms associated with larval metamorphosis in polychaetes. © 2011 Chandramouli et al; licensee BioMed Central Ltd.

Dynamic organization of genetic recombination proteins and chromosomes

International Nuclear Information System (INIS)

Essers, J.; Van Cappellen, G.; Van Drunen, E.; Theil, A.; Jaspers, N.N.G.J.; Houtsmuller, A.B.; Vermeulen, W.; Kanaar, R.

2003-01-01

Homologous recombination requires the co-ordinated action of the RAD52 group proteins, including Rad51, Rad52 and Rad54. Upon treatment of mammalian cells with ionizing radiation, these proteins accumulate into foci at sites of DSB induction. We probed the nature of the DNA damage-induced foci in living cells with the use of photobleaching techniques. These foci are not static assemblies of DNA repair proteins. Instead, they are dynamic structures of which Rad51 is a stable core component, while Rad52 and Rad54 reversibly interact with the structure. Furthermore, even though the RAD52 group proteins colocalize in the DNA damage-induced foci, the majority of the proteins are not part of the same multi-protein complex in the absence of DNA damage. Executing DNA transactions through dynamic multi-protein complexes, rather than stable holo-complexes, allows greater flexibility during the transaction. In case of DNA repair, for example, it allows cross talk between different DNA repair pathways and coupling to other DNA transactions, such as replication. In addition to the behavior of proteins in living cells, we have tracked chromosomes during cell division. Our results suggest that the relative position of chromosomes in the mother cell is conserved in its daughter cells

Dynamics of differentiation in magma reservoirs

Science.gov (United States)

Jaupart, Claude; Tait, Stephen

1995-09-01

In large magma chambers, gradients of temperature and composition develop due to cooling and to fractional crystallization. Unstable density differences lead to differential motions between melt and crystals, and a major goal is to explain how this might result in chemical differentiation of magma. Arriving at a full description of the physics of crystallizing magma chambers is a challenge because of the large number of processes potentially involved, the many coupled variables, and the different geometrical shapes. Furthermore, perturbations are caused by the reinjection of melt from a deep source, eruption to the Earth's surface, and the assimilation of country rock. Physical models of increasing complexity have been developed with emphasis on three fundamental approaches. One is, given that large gradients in temperature and composition may occur, to specify how to apply thermodynamic constraints so that coexisting liquid and solid compositions may be calculated. The second is to leave the differentiation trend as the solution to be found, i.e., to specify how cooling occurs and to predict the evolution of the composition of the residual liquid and of the solid forming. The third is to simplify the physics so that the effects of coupled heat and mass transfer may be studied with a reduced set of variables. The complex shapes of magma chambers imply that boundary layers develop with density gradients at various angles to gravity, leading to various convective flows and profiles qf liquid stratification. Early studies were mainly concerned with describing fluid flow in the liquid interior of large reservoirs, due to gradients developed at the margins. More recent work has focused on the internal structure and flow field of boundary layers and in particular on the gradients of solid fraction and interstitial melt composition which develop within them. Crystal settling may occur in a surprisingly diverse range of regimes and may lead to intermittent deposition

Acute differential effects of dietary protein quality on postprandial lipemia in obese non-diabetic subjects

DEFF Research Database (Denmark)

Holmer-Jensen, Jens; Mortensen, Lene Sundahl; Astrup, Arne

2013-01-01

Non-fasting triglyceridemia is much closer associated to cardiovascular risk compared to fasting triglyceridemia. We hypothesized that there would be acute differential effects of four common dietary proteins (cod protein, whey isolate, gluten, and casein) on postprandial lipemia in obese non......-diabetic subjects. To test the hypothesis we conducted a randomized, acute clinical intervention study with crossover design. We supplemented a fat rich mixed meal with one of four dietary proteins i.e. cod protein, whey protein, gluten or casein. Eleven obese non-diabetic subjects (age: 40-68, body mass index: 30...... concentration in the chylomicron rich fraction (P = .0293). Thus, we have demonstrated acute differential effects on postprandial metabolism of four dietary proteins supplemented to a fat rich mixed meal in obese non-diabetic subjects. Supplementation with whey protein caused lower postprandial lipemia compared...

Surface dynamics in allosteric regulation of protein-protein interactions: modulation of calmodulin functions by Ca2+.

Directory of Open Access Journals (Sweden)

Yosef Y Kuttner

2013-04-01

Full Text Available Knowledge of the structural basis of protein-protein interactions (PPI is of fundamental importance for understanding the organization and functioning of biological networks and advancing the design of therapeutics which target PPI. Allosteric modulators play an important role in regulating such interactions by binding at site(s orthogonal to the complex interface and altering the protein's propensity for complex formation. In this work, we apply an approach recently developed by us for analyzing protein surfaces based on steered molecular dynamics simulation (SMD to the study of the dynamic properties of functionally distinct conformations of a model protein, calmodulin (CaM, whose ability to interact with target proteins is regulated by the presence of the allosteric modulator Ca(2+. Calmodulin is a regulatory protein that acts as an intracellular Ca(2+ sensor to control a wide variety of cellular processes. We demonstrate that SMD analysis is capable of pinpointing CaM surfaces implicated in the recognition of both the allosteric modulator Ca(2+ and target proteins. Our analysis of changes in the dynamic properties of the CaM backbone elicited by Ca(2+ binding yielded new insights into the molecular mechanism of allosteric regulation of CaM-target interactions.

Differentially expressed genes in iron-induced prion protein conversion

International Nuclear Information System (INIS)

Kim, Minsun; Kim, Eun-hee; Choi, Bo-Ran; Woo, Hee-Jong

2016-01-01

The conversion of the cellular prion protein (PrP C ) to the protease-resistant isoform is the key event in chronic neurodegenerative diseases, including transmissible spongiform encephalopathies (TSEs). Increased iron in prion-related disease has been observed due to the prion protein-ferritin complex. Additionally, the accumulation and conversion of recombinant PrP (rPrP) is specifically derived from Fe(III) but not Fe(II). Fe(III)-mediated PK-resistant PrP (PrP res ) conversion occurs within a complex cellular environment rather than via direct contact between rPrP and Fe(III). In this study, differentially expressed genes correlated with prion degeneration by Fe(III) were identified using Affymetrix microarrays. Following Fe(III) treatment, 97 genes were differentially expressed, including 85 upregulated genes and 12 downregulated genes (≥1.5-fold change in expression). However, Fe(II) treatment produced moderate alterations in gene expression without inducing dramatic alterations in gene expression profiles. Moreover, functional grouping of identified genes indicated that the differentially regulated genes were highly associated with cell growth, cell maintenance, and intra- and extracellular transport. These findings showed that Fe(III) may influence the expression of genes involved in PrP folding by redox mechanisms. The identification of genes with altered expression patterns in neural cells may provide insights into PrP conversion mechanisms during the development and progression of prion-related diseases. - Highlights: • Differential genes correlated with prion degeneration by Fe(III) were identified. • Genes were identified in cell proliferation and intra- and extracellular transport. • In PrP degeneration, redox related genes were suggested. • Cbr2, Rsad2, Slc40a1, Amph and Mvd were expressed significantly.

Retinoblastoma protein functions as a molecular switch determining white versus brown adipocyte differentiation

DEFF Research Database (Denmark)

Hansen, Jacob B; Jørgensen, Claus; Petersen, Rasmus K

2004-01-01

Adipocyte precursor cells give raise to two major cell populations with different physiological roles: white and brown adipocytes. Here we demonstrate that the retinoblastoma protein (pRB) regulates white vs. brown adipocyte differentiation. Functional inactivation of pRB in wild-type mouse embryo...... fibroblasts (MEFs) and white preadipocytes by expression of simian virus 40 large T antigen results in the expression of the brown fat-specific uncoupling protein 1 (UCP-1) in the adipose state. Retinoblastoma gene-deficient (Rb-/-) MEFs and stem cells, but not the corresponding wild-type cells, differentiate...

Hindered protein dynamics in the presence of a cryoprotecting agent

Energy Technology Data Exchange (ETDEWEB)

Koeper, I. [Laboratoire Leon Brillouin, CEA Saclay, 91191 Gif-sur-Yvette (France); Physikdepartment E13, TU Muenchen, 85747 Garching (Germany); Bellissent-Funel, M.C. [Laboratoire Leon Brillouin, CEA Saclay, 91191 Gif-sur-Yvette (France)

2002-07-01

We present a study of the influence of trehalose, a well-known cryoprotecting disaccharide, on the dynamics of a protein, the C-phycocyanin. The dynamics is investigated in a time range from some picoseconds to several nanoseconds using different neutron-scattering techniques. Data obtained on samples containing hydrated powders of the protein in the presence of trehalose are compared to that of the protein alone, studied by neutron-scattering techniques as well as by molecular dynamics simulations. The analysis of time-of-flight data gives access to the geometry of the observed motions. These motions can be described via a model of a particle diffusing inside a sphere. We observe a slowing down of the movements of the protein due to the presence of trehalose of one to two orders of magnitude, while the geometry of the motions is conserved. (orig.)

Intermolecular detergent-membrane protein noes for the characterization of the dynamics of membrane protein-detergent complexes.

Science.gov (United States)

Eichmann, Cédric; Orts, Julien; Tzitzilonis, Christos; Vögeli, Beat; Smrt, Sean; Lorieau, Justin; Riek, Roland

2014-12-11

The interaction between membrane proteins and lipids or lipid mimetics such as detergents is key for the three-dimensional structure and dynamics of membrane proteins. In NMR-based structural studies of membrane proteins, qualitative analysis of intermolecular nuclear Overhauser enhancements (NOEs) or paramagnetic resonance enhancement are used in general to identify the transmembrane segments of a membrane protein. Here, we employed a quantitative characterization of intermolecular NOEs between (1)H of the detergent and (1)H(N) of (2)H-perdeuterated, (15)N-labeled α-helical membrane protein-detergent complexes following the exact NOE (eNOE) approach. Structural considerations suggest that these intermolecular NOEs should show a helical-wheel-type behavior along a transmembrane helix or a membrane-attached helix within a membrane protein as experimentally demonstrated for the complete influenza hemagglutinin fusion domain HAfp23. The partial absence of such a NOE pattern along the amino acid sequence as shown for a truncated variant of HAfp23 and for the Escherichia coli inner membrane protein YidH indicates the presence of large tertiary structure fluctuations such as an opening between helices or the presence of large rotational dynamics of the helices. Detergent-protein NOEs thus appear to be a straightforward probe for a qualitative characterization of structural and dynamical properties of membrane proteins embedded in detergent micelles.

Motion Tree Delineates Hierarchical Structure of Protein Dynamics Observed in Molecular Dynamics Simulation.

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Kei Moritsugu

Full Text Available Molecular dynamics (MD simulations of proteins provide important information to understand their functional mechanisms, which are, however, likely to be hidden behind their complicated motions with a wide range of spatial and temporal scales. A straightforward and intuitive analysis of protein dynamics observed in MD simulation trajectories is therefore of growing significance with the large increase in both the simulation time and system size. In this study, we propose a novel description of protein motions based on the hierarchical clustering of fluctuations in the inter-atomic distances calculated from an MD trajectory, which constructs a single tree diagram, named a "Motion Tree", to determine a set of rigid-domain pairs hierarchically along with associated inter-domain fluctuations. The method was first applied to the MD trajectory of substrate-free adenylate kinase to clarify the usefulness of the Motion Tree, which illustrated a clear-cut dynamics picture of the inter-domain motions involving the ATP/AMP lid and the core domain together with the associated amplitudes and correlations. The comparison of two Motion Trees calculated from MD simulations of ligand-free and -bound glutamine binding proteins clarified changes in inherent dynamics upon ligand binding appeared in both large domains and a small loop that stabilized ligand molecule. Another application to a huge protein, a multidrug ATP binding cassette (ABC transporter, captured significant increases of fluctuations upon binding a drug molecule observed in both large scale inter-subunit motions and a motion localized at a transmembrane helix, which may be a trigger to the subsequent structural change from inward-open to outward-open states to transport the drug molecule. These applications demonstrated the capabilities of Motion Trees to provide an at-a-glance view of various sizes of functional motions inherent in the complicated MD trajectory.

Potential biological process of X-linked inhibitor of apoptosis protein in renal cell carcinoma based upon differential protein expression analysis.

Science.gov (United States)

Chen, Chao; Zhao, Si Cong; Yang, Wen Zheng; Chen, Zong Ping; Yan, Yong

2018-01-01

The X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member of the IAP family and is a potent inhibitor of the caspase/apoptosis pathway. It has also been revealed that XIAP has additional biological functions that rely on its direct inhibition of apoptosis. In the present study, stably transfected Caki-1 cells with XIAP-knockdown were generated, and an isobaric tag for relative and absolute quantitation-based proteomics approach was employed to investigate the regulatory mechanism of XIAP in renal cell carcinoma (RCC). The results demonstrate that the sensitivity of the RCC cell line to apoptotic stimulation increased markedly with XIAP-knockdown. A number of differentially expressed proteins were detected between the original Caki-1 cell line and the XIAP-knockdown Caki-1 cell line; 87 at 0 h (prior to etoposide treatment), 178 at 0.5 h and 169 at 3 h, while no differentially expressed proteins were detected (ratio >1.5 or <0.5; P<0.05) at 12 h after etoposide treatment. Through analysis of the differentially expressed proteins, it was revealed that XIAP may participate in the tumor protein p53 pathway, the Wnt signaling pathway, glucose metabolism, endoplasmic reticulum stress, cytoskeletal regulation and DNA repair. These results indicate that XIAP may have a number of biological functions and may provide an insight into the biomedical significance of XIAP overexpression in RCC.

Group Dynamics and Individual Roles: A Differentiated Approach to Social-Emotional Learning

Science.gov (United States)

Dugas, Daryl

2017-01-01

Differentiated instruction is a set of strategies to help teachers meet each child where he or she is in order to improve students' engagement, lead them to do their best work, and maximize their success. This article describes a differentiated classroom management approach based in group dynamics which focuses on the development of group norms…

Nuclear translocation of the cytoskeleton-associated protein, smALP, upon induction of skeletal muscle differentiation

International Nuclear Information System (INIS)

Cambier, Linda; Pomies, Pascal

2011-01-01

Highlights: → The cytoskeleton-associated protein, smALP, is expressed in differentiated skeletal muscle. → smALP is translocated from the cytoplasm to the nucleus of C2C12 myoblasts upon induction of myogenesis. → The differentiation-dependent nuclear translocation of smALP occurs in parallel with the nuclear accumulation of myogenin. → The LIM domain of smALP is essential for the nuclear accumulation of the protein. → smALP might act in the nucleus to control some critical aspect of the muscle differentiation process. -- Abstract: The skALP isoform has been shown to play a critical role in actin organization and anchorage within the Z-discs of skeletal muscles, but no data is available on the function of the smALP isoform in skeletal muscle cells. Here, we show that upon induction of differentiation a nuclear translocation of smALP from the cytoplasm to the nucleus of C2C12 myoblasts, concomitant to an up-regulation of the protein expression, occurs in parallel with the nuclear accumulation of myogenin. Moreover, we demonstrate that the LIM domain of smALP is essential for the nuclear translocation of the protein.

Methyl CpG–binding proteins induce large-scale chromatin reorganization during terminal differentiation

Science.gov (United States)

Brero, Alessandro; Easwaran, Hariharan P.; Nowak, Danny; Grunewald, Ingrid; Cremer, Thomas; Leonhardt, Heinrich; Cardoso, M. Cristina

2005-01-01

Pericentric heterochromatin plays an important role in epigenetic gene regulation. We show that pericentric heterochromatin aggregates during myogenic differentiation. This clustering leads to the formation of large chromocenters and correlates with increased levels of the methyl CpG–binding protein MeCP2 and pericentric DNA methylation. Ectopic expression of fluorescently tagged MeCP2 mimicked this effect, causing a dose-dependent clustering of chromocenters in the absence of differentiation. MeCP2-induced rearrangement of heterochromatin occurred throughout interphase, did not depend on the H3K9 histone methylation pathway, and required the methyl CpG–binding domain (MBD) only. Similar to MeCP2, another methyl CpG–binding protein, MBD2, also increased during myogenic differentiation and could induce clustering of pericentric regions, arguing for functional redundancy. This MeCP2- and MBD2-mediated chromatin reorganization may thus represent a molecular link between nuclear genome topology and the epigenetic maintenance of cellular differentiation. PMID:15939760

Schisandrae fructus enhances myogenic differentiation and inhibits atrophy through protein synthesis in human myotubes

Directory of Open Access Journals (Sweden)

Kim CH

2016-05-01

Full Text Available Cy Hyun Kim,1,2,* Jin-Hong Shin,1,3,* Sung Jun Hwang,1,2 Yung Hyun Choi,4 Dae-Seong Kim,1,3 Cheol Min Kim2,51Research Institute of Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan, 2Center for Anti-Aging Industry, Pusan National University, Busan, 3Department of Neurology, Pusan National University Yangsan Hospital, Yangsan, 4Department of Biochemistry, Dong-eui University College of Korean Medicine, Busan, 5Department of Biomedical Informatics, Pusan National University School of Medicine, Yangsan, Republic of Korea*These authors contributed equally to this work Abstract: Schisandrae fructus (SF has recently been reported to increase skeletal muscle mass and inhibit atrophy in mice. We investigated the effect of SF extract on human myotube differentiation and its acting pathway. Various concentrations (0.1–10 µg/mL of SF extract were applied on human skeletal muscle cells in vitro. Myotube area and fusion index were measured to quantify myotube differentiation. The maximum effect was observed at 0.5 µg/mL of SF extract, enhancing differentiation up to 1.4-fold in fusion index and 1.6-fold in myotube area at 8 days after induction of differentiation compared to control. Phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 and 70 kDa ribosomal protein S6 kinase, which initiate translation as downstream of mammalian target of rapamycin pathway, was upregulated in early phases of differentiation after SF treatment. SF also attenuated dexamethasone-induced atrophy. In conclusion, we show that SF augments myogenic differentiation and attenuates atrophy by increasing protein synthesis through mammalian target of rapamycin/70 kDa ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1 signaling pathway in human myotubes. SF can be a useful natural dietary supplement in increasing skeletal muscle mass, especially in the aged

Molecular nonlinear dynamics and protein thermal uncertainty quantification

Science.gov (United States)

Xia, Kelin; Wei, Guo-Wei

2014-01-01

This work introduces molecular nonlinear dynamics (MND) as a new approach for describing protein folding and aggregation. By using a mode system, we show that the MND of disordered proteins is chaotic while that of folded proteins exhibits intrinsically low dimensional manifolds (ILDMs). The stability of ILDMs is found to strongly correlate with protein energies. We propose a novel method for protein thermal uncertainty quantification based on persistently invariant ILDMs. Extensive comparison with experimental data and the state-of-the-art methods in the field validate the proposed new method for protein B-factor prediction. PMID:24697365

Microsecond molecular dynamics simulation shows effect of slow loop dynamics on backbone amide order parameters of proteins

DEFF Research Database (Denmark)

Maragakis, Paul; Lindorff-Larsen, Kresten; Eastwood, Michael P

2008-01-01

. Molecular dynamics (MD) simulation provides a complementary approach to the study of protein dynamics on similar time scales. Comparisons between NMR spectroscopy and MD simulations can be used to interpret experimental results and to improve the quality of simulation-related force fields and integration......A molecular-level understanding of the function of a protein requires knowledge of both its structural and dynamic properties. NMR spectroscopy allows the measurement of generalized order parameters that provide an atomistic description of picosecond and nanosecond fluctuations in protein structure...... methods. However, apparent systematic discrepancies between order parameters extracted from simulations and experiments are common, particularly for elements of noncanonical secondary structure. In this paper, results from a 1.2 micros explicit solvent MD simulation of the protein ubiquitin are compared...

Differential regulation of genomic imprinting by TET proteins in embryonic stem cells.

Science.gov (United States)

Liu, Lizhi; Mao, Shi-Qing; Ray, Chelsea; Zhang, Yu; Bell, Fong T; Ng, Sheau-Fang; Xu, Guo-Liang; Li, Xiajun

2015-09-01

TET proteins have been found to play an important role in active demethylation at CpG sites in mammals. There are some reports implicating their functions in removal of DNA methylation imprint at the imprinted regions in the germline. However, it is not well established whether TET proteins can also be involved in demethylation of DNA methylation imprint in embryonic stem (ES) cells. Here we report that loss of TET proteins caused a significant increase in DNA methylation at the Igf2-H19 imprinted region in ES cells. We also observed a variable increase in DNA methylation at the Peg1 imprinted region in the ES clones devoid of TET proteins, in particular in the differentiated ES cells. By contrast, we did not observe a significant increase of DNA methylation imprint at the Peg3, Snrpn and Dlk1-Dio3 imprinted regions in ES cells lacking TET proteins. Interestingly, loss of TET proteins did not result in a significant increase of DNA methylation imprint at the Igf2-H19 and Peg1 imprinted regions in the embryoid bodies (EB). Therefore, TET proteins seem to be differentially involved in maintaining DNA methylation imprint at a subset of imprinted regions in ES cells and EBs. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Exploration of Protein Conformational Change with PELE and Meta-Dynamics.

Science.gov (United States)

Cossins, Benjamin P; Hosseini, Ali; Guallar, Victor

2012-03-13

Atomistic molecular simulation methods are now able to explore complex protein or protein-ligand dynamical space in a tractable way with methods such as meta-dynamics or adaptive biasing force. However, many of these methods either require a careful selection of reaction coordinates or the knowledge of an initial pathway of some kind. Thus, it is important that effective methods are developed to produce this pathway data in an efficient fashion. PELE, a proven protein-ligand sampling code, has been developed to provide rapid protein sampling in highly flexible cases, using a reduced network model eigen problem approach. The resulting method is able to rapidly sample configuration space with very general driving information. When applied to ubiquitin, PELE was able to reproduce RMSD and average force data found in molecular dynamics simulations. PELE was also applied to explore the opening/closing transition of T4 lysozyme. A meta-dynamics exploration using a low energy pathway validated that the configurations explored by PELE represent the most populated regions of phase space. PELE and meta-dynamics explorations also discovered a low free energy region where a large cross-domain helix of T4 lysozyme is broken in two. There is previous NMR evidence for the validity of this unfolded helix region.

Neutron scattering studies on protein dynamics using the human myelin peripheral membrane protein P2

Directory of Open Access Journals (Sweden)

Laulumaa Saara

2015-01-01

Full Text Available Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect protein dynamics, and in the case of P2, they may explain functional differences between the two proteins.

Neutron scattering studies on protein dynamics using the human myelin peripheral membrane protein P2

Science.gov (United States)

Laulumaa, Saara; Kursula, Petri; Natali, Francesca

2015-01-01

Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect protein dynamics, and in the case of P2, they may explain functional differences between the two proteins.

The Dynamics of Gender Earnings Differentials: Evidence from Establishment Data

OpenAIRE

Barth, Erling; Pekkala Kerr, Sari; Olivetti, Claudia

2017-01-01

We use a unique match between the 2000 Decennial Census of the United States and the Longitudinal Employer Household Dynamics (LEHD) data to analyze how much of the increase in the gender earnings gap over the lifecycle comes from shifts in the sorting of men and women across high- and low-pay establishments and how much is due to differential earnings growth within establishments. We find that for the college educated the increase is substantial and, for the most part, due to differential ea...

A vesicle trafficking protein αSNAP regulates Paneth cell differentiation in vivo.

Science.gov (United States)

Lechuga, Susana; Naydenov, Nayden G; Feygin, Alex; Jimenez, Antonio J; Ivanov, Andrei I

2017-05-13

A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Activation of peroxisome proliferator-activated receptor gamma bypasses the function of the retinoblastoma protein in adipocyte differentiation

DEFF Research Database (Denmark)

Hansen, Jacob B.; Petersen, R K; Larsen, B M

1999-01-01

The retinoblastoma protein (pRB) is an important regulator of development, proliferation, and cellular differentiation. pRB was recently shown to play a pivotal role in adipocyte differentiation, to interact physically with adipogenic CCAAT/enhancer-binding proteins (C/EBPs), and to positively...

Degradation of protein translation machinery by amino acid starvation-induced macroautophagy

DEFF Research Database (Denmark)

Gretzmeier, Christine; Eiselein, Sven; Johnson, Gregory R.

2017-01-01

, unbiased approaches relying on quantitative mass spectrometry-based proteomics. Macroautophagy is induced by rapamycin treatment, and by amino acid and glucose starvation in differentially, metabolically labeled cells. Protein dynamics are linked to image-based models of autophagosome turnover. Depending...... on the inducing stimulus, protein as well as organelle turnover differ. Amino acid starvation-induced macroautophagy leads to selective degradation of proteins important for protein translation. Thus, protein dynamics reflect cellular conditions in the respective treatment indicating stimulus-specific pathways...

Novel leukocyte protein, Trojan, differentially expressed during thymocyte development.

Science.gov (United States)

Petrov, Petar; Motobu, Maki; Salmi, Jussi; Uchida, Tatsuya; Vainio, Olli

2010-04-01

"Trojan" is a novel cell surface protein, discovered from chicken embryonic thymocytes on the purpose to identify molecules involved in T cell differentiation. The molecule is predicted as a type I transmembrane protein having a Sushi and two fibronectin type III domains and a pair of intracellular phosphorylation sites. Its transcript expression is specific for lymphoid tissues and the presence of the protein on the surface of recirculating lymphocytes and macrophages was confirmed by immunofluorescence analysis. In thymus, about half of the double negative (CD4(-) CD8(-)) and CD8 single positive and the majority of CD4 single positive cells express Trojan with a relatively high intensity. However, only a minority of the double positive (CD4(+) CD8(+)) cells are positive for Trojan. This expression pattern, similar to that of some proteins with anti-apoptotic and function, like IL-7Ralpha, makes Trojan an attractive candidate of having an anti-apoptotic role. Copyright 2010 Elsevier Ltd. All rights reserved.

Enhancing protein adsorption simulations by using accelerated molecular dynamics.

Directory of Open Access Journals (Sweden)

Christian Mücksch

Full Text Available The atomistic modeling of protein adsorption on surfaces is hampered by the different time scales of the simulation ([Formula: see text][Formula: see text]s and experiment (up to hours, and the accordingly different 'final' adsorption conformations. We provide evidence that the method of accelerated molecular dynamics is an efficient tool to obtain equilibrated adsorption states. As a model system we study the adsorption of the protein BMP-2 on graphite in an explicit salt water environment. We demonstrate that due to the considerably improved sampling of conformational space, accelerated molecular dynamics allows to observe the complete unfolding and spreading of the protein on the hydrophobic graphite surface. This result is in agreement with the general finding of protein denaturation upon contact with hydrophobic surfaces.

Molecular Effects of Concentrated Solutes on Protein Hydration, Dynamics, and Electrostatics.

Science.gov (United States)

Abriata, Luciano A; Spiga, Enrico; Peraro, Matteo Dal

2016-08-23

Most studies of protein structure and function are performed in dilute conditions, but proteins typically experience high solute concentrations in their physiological scenarios and biotechnological applications. High solute concentrations have well-known effects on coarse protein traits like stability, diffusion, and shape, but likely also perturb other traits through finer effects pertinent at the residue and atomic levels. Here, NMR and molecular dynamics investigations on ubiquitin disclose variable interactions with concentrated solutes that lead to localized perturbations of the protein's surface, hydration, electrostatics, and dynamics, all dependent on solute size and chemical properties. Most strikingly, small polar uncharged molecules are sticky on the protein surface, whereas charged small molecules are not, but the latter still perturb the internal protein electrostatics as they diffuse nearby. Meanwhile, interactions with macromolecular crowders are favored mainly through hydrophobic, but not through polar, surface patches. All the tested small solutes strongly slow down water exchange at the protein surface, whereas macromolecular crowders do not exert such strong perturbation. Finally, molecular dynamics simulations predict that unspecific interactions slow down microsecond- to millisecond-timescale protein dynamics despite having only mild effects on pico- to nanosecond fluctuations as corroborated by NMR. We discuss our results in the light of recent advances in understanding proteins inside living cells, focusing on the physical chemistry of quinary structure and cellular organization, and we reinforce the idea that proteins should be studied in native-like media to achieve a faithful description of their function. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

[Screening differentially expressed plasma proteins in cold stress rats based on iTRAQ combined with mass spectrometry technology].

Science.gov (United States)

Liu, Yan-zhi; Guo, Jing-ru; Peng, Meng-ling; Ma, Li; Zhen, Li; Ji, Hong; Yang, Huan-min

2015-09-01

Isobaric tags for relative and absolute quantitation (iTRAQ) combined with mass spectrometry were used to screen differentially expressed plasma proteins in cold stress rats. Thirty health SPF Wistar rats were randomly divided into cold stress group A and control group B, then A and B were randomly divided into 3 groups (n = 5): A1, A2, A3 and B1, B2, B3. The temperature of room raising was (24.0 +/- 0.1) degrees C, and the cold stress temperature was (4.0 +/- 0.1) degrees C. The rats were treated with different temperatures until 12 h. The abdominal aortic blood was collected with heparin anticoagulation suction tube. Then, the plasma was separated for protein extraction, quantitative, enzymolysis, iTHAQ labeling, scx fractionation and mass spectrometry analysis. Totally, 1085 proteins were identified in the test, 39 differentially expressed proteins were screened, including 29 up-regulated proteins and 10 down-regulated proteins. Three important differentially expressed proteins related to cold stress were screened by bioinfonnatics analysis (Minor histocompatihility protein HA-1, Has-related protein Rap-1b, Integrin beta-1). In the experiment, the differentially expressed plasma proteins were successfully screened in cold stress rats. iTRAQ technology provided a good platform to screen protein diaguostic markers on cold stress rats, and laid a good foundation for further. study on animal cold stress mechanism.

Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

Directory of Open Access Journals (Sweden)

Alberto Miranda

2011-04-01

Full Text Available Cellular prion protein (PRNP is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs. Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB differentiation in mouse Prnp-null (KO and WT embryonic stem cell (ESC lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5 in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel and SPRN (Shadoo, whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

The dynamic multisite interactions between two intrinsically disordered proteins

KAUST Repository

Wu, Shaowen; Wang, Dongdong; Liu, Jin; Feng, Yitao; Weng, Jingwei; Li, Yu; Gao, Xin; Liu, Jianwei; Wang, Wenning

2017-01-01

Protein interactions involving intrinsically disordered proteins (IDPs) comprise a variety of binding modes, from the well characterized folding upon binding to dynamic fuzzy complex. To date, most studies concern the binding of an IDP to a

Differentiation between benign and malignant breast lesions using fat-suppressed dynamic MR imaging

International Nuclear Information System (INIS)

Koshiishi, Takeshi; Isomoto, Ichirou; Nakamura, Kazukuni; Kajiwara, Yoshifumi; Izawa, Kunihide

1998-01-01

To assess the value and problems of fat-suppressed dynamic MR imaging in differentiating between benign and malignant lesions. In twenty-nine patients who underwent excisional biopsy or surgical resection, fat-suppressed dynamic MR imaging was performed with a 0.5 T superconducting magnet. Pre- and post-contrast 3D-spoiled gradient echo sequences were employed with fat suppression. We calculated and evaluated the contrast-to-noise ratio (CNR) and contrast enhancement ratio (CER) at each contrast determination time (CDT), which is the intermediate time in the scan. Time intensity curves of CNR showed no statistically significant difference between cancers and other benign lesions. The difference in CER between malignant and benign disease was highly significant (p=0.006) at CDT 45 sec., but there was great overlap in the time intensity curve of CER after CDT 45 sec. When we attempt to differentiate malignant from benign breast lesions by dynamic MR imaging, comparison of CNR is impertinent, and we should evaluate the differential diagnosis of cancer versus benign lesions by means of CER at CDT points of about 45 sec. (author)

Identifying protein complex by integrating characteristic of core-attachment into dynamic PPI network.

Directory of Open Access Journals (Sweden)

Xianjun Shen

Full Text Available How to identify protein complex is an important and challenging task in proteomics. It would make great contribution to our knowledge of molecular mechanism in cell life activities. However, the inherent organization and dynamic characteristic of cell system have rarely been incorporated into the existing algorithms for detecting protein complexes because of the limitation of protein-protein interaction (PPI data produced by high throughput techniques. The availability of time course gene expression profile enables us to uncover the dynamics of molecular networks and improve the detection of protein complexes. In order to achieve this goal, this paper proposes a novel algorithm DCA (Dynamic Core-Attachment. It detects protein-complex core comprising of continually expressed and highly connected proteins in dynamic PPI network, and then the protein complex is formed by including the attachments with high adhesion into the core. The integration of core-attachment feature into the dynamic PPI network is responsible for the superiority of our algorithm. DCA has been applied on two different yeast dynamic PPI networks and the experimental results show that it performs significantly better than the state-of-the-art techniques in terms of prediction accuracy, hF-measure and statistical significance in biology. In addition, the identified complexes with strong biological significance provide potential candidate complexes for biologists to validate.

Differential abundance of egg white proteins in laying hens treated with corticosterone.

Science.gov (United States)

Kim, Jimin; Choi, Yang-Ho

2014-12-24

Stressful environments can affect not only egg production and quality but also gene and protein abundance in the ovary and oviduct in laying hens. The oviductal magnum of laying hens is the organ responsible for the synthesis and secretion of egg white proteins. The objective of this study was to investigate the effects of dietary corticosterone as a stress model on the abundance of proteins in the egg white and of mRNA and proteins in the magnum in laying hens. After a 14-day acclimation, 40 laying hens were divided into two groups which were provided for the next 14 days with either control (Control) or corticosterone (Stress) diet containing at 30 mg/kg. Corticosterone treatment resulted in increased feed intake (P ≤ 0.05) and decreased egg production. Two-dimensional electrophoresis (2DE) with MALDI-TOF/TOF MS/MS using eggs obtained on days 0 and 5 revealed differential abundance of egg white proteins by Stress: transiently expressed in neural precursors (TENP), hemopexin (HPX), IgY-Fcυ3-4, and extracellular fatty acid-binding protein (Ex-FABP) were decreased while ovoinhibitor and ovalbumin-related protein X (OVAX) were increased on days 5 vs 0 (P ≤ 0.05). Expression of mRNAs and proteins was also significantly modulated in the magnum of hens in Stress on day 14 (P ≤ 0.05). In conclusion, the current study provides the first evidence showing that dietary corticosterone modulates protein abundance in the egg white in laying hens, and it suggests that environmental stress can differentially modify expression of egg white proteins in laying hens.

Markov dynamic models for long-timescale protein motion.

KAUST Repository

Chiang, Tsung-Han

2010-06-01

Molecular dynamics (MD) simulation is a well-established method for studying protein motion at the atomic scale. However, it is computationally intensive and generates massive amounts of data. One way of addressing the dual challenges of computation efficiency and data analysis is to construct simplified models of long-timescale protein motion from MD simulation data. In this direction, we propose to use Markov models with hidden states, in which the Markovian states represent potentially overlapping probabilistic distributions over protein conformations. We also propose a principled criterion for evaluating the quality of a model by its ability to predict long-timescale protein motions. Our method was tested on 2D synthetic energy landscapes and two extensively studied peptides, alanine dipeptide and the villin headpiece subdomain (HP-35 NleNle). One interesting finding is that although a widely accepted model of alanine dipeptide contains six states, a simpler model with only three states is equally good for predicting long-timescale motions. We also used the constructed Markov models to estimate important kinetic and dynamic quantities for protein folding, in particular, mean first-passage time. The results are consistent with available experimental measurements.

Markov dynamic models for long-timescale protein motion.

KAUST Repository

Chiang, Tsung-Han; Hsu, David; Latombe, Jean-Claude

2010-01-01

Molecular dynamics (MD) simulation is a well-established method for studying protein motion at the atomic scale. However, it is computationally intensive and generates massive amounts of data. One way of addressing the dual challenges of computation efficiency and data analysis is to construct simplified models of long-timescale protein motion from MD simulation data. In this direction, we propose to use Markov models with hidden states, in which the Markovian states represent potentially overlapping probabilistic distributions over protein conformations. We also propose a principled criterion for evaluating the quality of a model by its ability to predict long-timescale protein motions. Our method was tested on 2D synthetic energy landscapes and two extensively studied peptides, alanine dipeptide and the villin headpiece subdomain (HP-35 NleNle). One interesting finding is that although a widely accepted model of alanine dipeptide contains six states, a simpler model with only three states is equally good for predicting long-timescale motions. We also used the constructed Markov models to estimate important kinetic and dynamic quantities for protein folding, in particular, mean first-passage time. The results are consistent with available experimental measurements.

Selective solubilization of membrane proteins differentially labeled by p-chloromercuribenzenesulfonic acid in the presence of sucrose

International Nuclear Information System (INIS)

M'Batchi, B.; Pichelin, D.; Delrot, S.

1987-01-01

Broadbean (Vicia faba L.) leaf discs have been incubated with the slowly permeant thiol reagent [ 203 Hg]-para-chloromercuribenzenesulfonic acid (PCMBS) in the presence or in the absence of sucrose, and the release of PCMBS-labeled proteins has been monitored in media containing various concentrations of urea, ethylene glycol-bis-(β-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA), sodium cholate, sodium dodecyl sulfate, Triton X-100, octylglucoside or (3-[3-cholamidopropyl)-dimethylammonio] 1-propane-sulfonate)(CHAPS). The proteins differentially labeled by PCMBS in the presence of sucrose which, on the basis of previous results, are assumed to included the sucrose carrier, were preferentially solubilized by 1% CHAPS, 1% octylglucoside, or 1% Triton X-100. Other PCMBS-labeled proteins (background proteins) could be partially removed by EGTA, urea, or 0.1% cholate. Sequential treatment by 10 mM EGTA and 1% CHAPS was found to give a fraction highly enriched in the differentially labeled proteins. Analysis of the specific activity of microsomal pellets suggests that the results obtained with leaf discs give a good account of what is occurring at the plasma membrane level. These data, which suggest that the proteins differentially labeled, by PCMBS in the presence of sucrose are intrinsic membrane proteins, can be used to solubilize these proteins from microsomal fractions

msiDBN: A Method of Identifying Critical Proteins in Dynamic PPI Networks

Directory of Open Access Journals (Sweden)

Yuan Zhang

2014-01-01

Full Text Available Dynamics of protein-protein interactions (PPIs reveals the recondite principles of biological processes inside a cell. Shown in a wealth of study, just a small group of proteins, rather than the majority, play more essential roles at crucial points of biological processes. This present work focuses on identifying these critical proteins exhibiting dramatic structural changes in dynamic PPI networks. First, a comprehensive way of modeling the dynamic PPIs is presented which simultaneously analyzes the activity of proteins and assembles the dynamic coregulation correlation between proteins at each time point. Second, a novel method is proposed, named msiDBN, which models a common representation of multiple PPI networks using a deep belief network framework and analyzes the reconstruction errors and the variabilities across the time courses in the biological process. Experiments were implemented on data of yeast cell cycles. We evaluated our network construction method by comparing the functional representations of the derived networks with two other traditional construction methods. The ranking results of critical proteins in msiDBN were compared with the results from the baseline methods. The results of comparison showed that msiDBN had better reconstruction rate and identified more proteins of critical value to yeast cell cycle process.

Is a malleable protein necessarily highly dynamic?

DEFF Research Database (Denmark)

Kjærgaard, Magnus; Poulsen, Flemming Martin; Teilum, Kaare

2012-01-01

core of NCBD in the ligand-free state and in a well-folded complex with the ligand activator for thyroid hormone and retinoid receptors using multiple NMR methods including methyl chemical shifts, coupling constants, and methyl order parameters. From all NMR measures, the aliphatic side chains...... in the hydrophobic core are slightly more dynamic in the free protein than in the complex, but have mobility comparable to the hydrophobic cores of average folded proteins. Urea titration monitored by NMR reveals that all parts of the protein, including the side-chain packing in the hydrophobic core, denatures...

Effects of low dose radiation on differential expression of serum protein in mice

International Nuclear Information System (INIS)

Chen Wei; He Ying; Shen Xianrong

2014-01-01

The aim is to find out the key proteins related with low dose radiation (Ld) by parametric technology, which provided the theory foundation for LDR protection Two-dimensional electrophoresis (2-DE) was performed on serum protein Differential expression proteins were identified by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and database analysis Compared with the control group, 7 altered proteins was definite in terms of apolipoprotein C-Ⅲ, beta-globin parotid secretory protein alpha-2-macroglobulin precursor, mouse transthyretin, C1qc protein and clusterin. Some proteins related with LDR are found. It may provide some new explanations for the mechanism of LDR. (authors)

Solution structure and dynamics of melanoma inhibitory activity protein

International Nuclear Information System (INIS)

Lougheed, Julie C.; Domaille, Peter J.; Handel, Tracy M.

2002-01-01

Melanoma inhibitory activity (MIA) is a small secreted protein that is implicated in cartilage cell maintenance and melanoma metastasis. It is representative of a recently discovered family of proteins that contain a Src Homologous 3 (SH3) subdomain. While SH3 domains are normally found in intracellular proteins and mediate protein-protein interactions via recognition of polyproline helices, MIA is single-domain extracellular protein, and it probably binds to a different class of ligands.Here we report the assignments, solution structure, and dynamics of human MIA determined by heteronuclear NMR methods. The structures were calculated in a semi-automated manner without manual assignment of NOE crosspeaks, and have a backbone rmsd of 0.38 A over the ordered regions of the protein. The structure consists of an SH3-like subdomain with N- and C-terminal extensions of approximately 20 amino acids each that together form a novel fold. The rmsd between the solution structure and our recently reported crystal structure is 0.86 A over the ordered regions of the backbone, and the main differences are localized to the most dynamic regions of the protein. The similarity between the NMR and crystal structures supports the use of automated NOE assignments and ambiguous restraints to accelerate the calculation of NMR structures

Conformational dynamics of a protein in the folded and the unfolded state

Energy Technology Data Exchange (ETDEWEB)

Fitter, Joerg

2003-08-01

In a quasielastic neutron scattering experiment, the picosecond dynamics of {alpha}-amylase was investigated for the folded and the unfolded state of the protein. In order to ensure a reasonable interpretation of the internal protein dynamics, the protein was measured in D{sub 2}O-buffer solution. The much higher structural flexibility of the pH induced unfolded state as compared to the native folded state was quantified using a simple analytical model, describing a local diffusion inside a sphere. In terms of this model the conformational volume, which is explored mainly by confined protein side-chain movements, is parameterized by the radius of a sphere (folded state, r=1.2 A; unfolded state, 1.8 A). Differences in conformational dynamics between the folded and the unfolded state of a protein are of fundamental interest in the field of protein science, because they are assumed to play an important role for the thermodynamics of folding/unfolding transition and for protein stability.

Dynamics of proteins and of their hydration layer studied by neutron scattering and additional biophysical methods

International Nuclear Information System (INIS)

Gallat, Francois-Xavier

2011-01-01

This thesis work focused on the dynamics of proteins, surrounded by their hydration layer, a water shell around the protein vital for its biological function. Each of these components is accompanied by a specific dynamics which union reforms the complex energy landscape of the system. The joint implementation of selective deuteration, incoherent neutron scattering and tera-hertz spectroscopy allowed to explore the dynamics of proteins and that of the hydration shell. The influence of the folding state of protein on its dynamics has been studied by elastic neutron scattering. Globular proteins were less dynamic than its intrinsically disordered analogues. Themselves appear to be stiffer than non-physiological unfolded proteins. The oligomerization state and the consequences on the dynamics were investigated. Aggregates of a globular protein proved to be more flexible than the soluble form. In contrast, aggregates of a disordered protein showed lower average dynamics compared to the soluble form. These observations demonstrate the wide range of dynamics among the proteome. Incoherent neutron scattering experiences on the hydration layer of globular and disordered proteins have yielded information on the nature of water motion around these proteins. The measurements revealed the presence of translational motions concomitant with the onset of the transition dynamics of hydration layers, at 220 K. Measurements have also shown a stronger coupling between a disordered protein and its hydration water, compared to a globular protein and its hydration shell. The nature of the hydration layer and its influence on its dynamics has been explored with the use of polymers that mimic the water behavior and that act as a source of flexibility for the protein. Eventually, the dynamics of methyl groups involved in the dynamical changes observed at 150 and 220 K, was investigated. (author) [fr

Differential protein modulation in midguts of Aedes aegypti infected with chikungunya and dengue 2 viruses.

Science.gov (United States)

Tchankouo-Nguetcheu, Stéphane; Khun, Huot; Pincet, Laurence; Roux, Pascal; Bahut, Muriel; Huerre, Michel; Guette, Catherine; Choumet, Valérie

2010-10-05

Arthropod borne virus infections cause several emerging and resurgent infectious diseases. Among the diseases caused by arboviruses, dengue and chikungunya are responsible for a high rate of severe human diseases worldwide. The midgut of mosquitoes is the first barrier for pathogen transmission and is a target organ where arboviruses must replicate prior to infecting other organs. A proteomic approach was undertaken to characterize the key virus/vector interactions and host protein modifications that happen in the midgut for viral transmission to eventually take place. Using a proteomics differential approach with two-Dimensional Differential in-Gel Electrophoresis (2D-DIGE), we defined the protein modulations in the midgut of Aedes aegypti that were triggered seven days after an oral infection (7 DPI) with dengue 2 (DENV-2) and chikungunya (CHIKV) viruses. Gel profile comparisons showed that the level of 18 proteins was modulated by DENV-2 only and 12 proteins were modulated by CHIKV only. Twenty proteins were regulated by both viruses in either similar or different ways. Both viruses caused an increase of proteins involved in the generation of reactive oxygen species, energy production, and carbohydrate and lipid metabolism. Midgut infection by DENV-2 and CHIKV triggered an antioxidant response. CHIKV infection produced an increase of proteins involved in detoxification. Our study constitutes the first analysis of the protein response of Aedes aegypti's midgut infected with viruses belonging to different families. It shows that the differentially regulated proteins in response to viral infection include structural, redox, regulatory proteins, and enzymes for several metabolic pathways. Some of these proteins like antioxidant are probably involved in cell protection. On the other hand, we propose that the modulation of other proteins like transferrin, hsp60 and alpha glucosidase, may favour virus survival, replication and transmission, suggesting a subversion of

Differentially expressed proteins on postoperative 3 days healing in rabbit Achilles tendon rupture model after early kinesitherapy.

Science.gov (United States)

Jialili, Ainuer; Jielile, Jiasharete; Abudoureyimu, Shajidan; Sabirhazi, Gulnur; Redati, Darebai; Bai, Jing-Ping; Bin, Liang; Duisabai, Sailike; Aishan, Jiangaguli; Kasimu, Haxiaobieke

2011-04-01

Surgical repair of Achilles tendon (AT) rupture should immediately be followed by active tendon mobilization. The optimal time as to when the mobilization should begin is important yet controversial. Early kinesitherapy leads to reduced rehabilitation period. However, an insight into the detailed mechanism of this process has not been gained. Proteomic technique can be used to separate and purify the proteins by differential expression profile which is related to the function of different proteins, but research in the area of proteomic analysis of AT 3 days after repair has not been studied so far. Forty-seven New Zealand white rabbits were randomized into 3 groups. Group A (immobilization group, n equal to 16) received postoperative cast immobilization; Group B (early motion group, n equal to 16) received early active motion treatments immediately following the repair of AT rupture from tenotomy. Another 15 rabbits served as control group (Group C). The AT samples were prepared 3 days following the microsurgery. The proteins were separated employing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). PDQuest software version 8.0 was used to identify differentially expressed proteins, followed by peptide mass fingerprint (PMF) and tandem mass spectrum analysis, using the National Center for Biotechnology Information (NCBI) protein database retrieval and then for bioinformatics analysis. A mean of 446.33, 436.33 and 462.67 protein spots on Achilles tendon samples of 13 rabbits in Group A, 14 rabbits in Group B and 13 rabbits in Group C were successfully detected in the 2D-PAGE. There were 40, 36 and 79 unique proteins in Groups A, B and C respectively. Some differentially expressed proteins were enzyme with the gel, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We successfully identified 9 and 11 different proteins in Groups A and B, such as GAPDH, phosphoglycerate kinase 1, pro-alpha-1 type 1 collagen

An Analysis on a Dynamic Amplifier and Calibration Methods for a Pseudo-Differential Dynamic Comparator

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Paik, Daehwa; Miyahara, Masaya; Matsuzawa, Akira

This paper analyzes a pseudo-differential dynamic comparator with a dynamic pre-amplifier. The transient gain of a dynamic pre-amplifier is derived and applied to equations of the thermal noise and the regeneration time of a comparator. This analysis enhances understanding of the roles of transistor's parameters in pre-amplifier's gain. Based on the calculated gain, two calibration methods are also analyzed. One is calibration of a load capacitance and the other is calibration of a bypass current. The analysis helps designers' estimation for the accuracy of calibration, dead-zone of a comparator with a calibration circuit, and the influence of PVT variation. The analyzed comparator uses 90-nm CMOS technology as an example and each estimation is compared with simulation results.

Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

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Yuping Ren

2017-12-01

Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in pediatric populations globally. This study examined proteomic profile changes in A549 cells infected with hMPV and two attenuated mutants with deleted PDZ domain-binding motif(s in the M2-2 protein. These motifs are involved in the interruption of antiviral signaling, namely the interaction between the TNF receptor associated factor (TRAF and mitochondrial antiviral-signaling (MAVS proteins. The aim of this study was to provide insight into the overall and novel impact of M2-2 motifs on cellular responses via an unbiased comparison. Tandem mass tagging, stable isotope labeling, and high-resolution mass spectrometry were used for quantitative proteomic analysis. Using quantitative proteomics and Venn analysis, 1248 common proteins were detected in all infected samples of both technical sets. Hierarchical clustering of the differentiated proteome displayed distinct proteomic signatures that were controlled by the motif(s. Bioinformatics and experimental analysis confirmed the differentiated proteomes, revealed novel cellular biological events, and implicated key pathways controlled by hMPV M2-2 PDZ domain-binding motif(s. This provides further insight for evaluating M2-2 mutants as potent vaccine candidates.

Digestion of cooked meat proteins is slightly affected by age as assessed using the dynamic gastrointestinal TIM model and mass spectrometry.

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Denis, S; Sayd, T; Georges, A; Chambon, C; Chalancon, S; Santé-Lhoutellier, V; Blanquet-Diot, S

2016-06-15

In humans, meat ensures the supply of proteins with high nutritional value and indispensable amino acids. The main goal of the present study was to compare the degradation of meat proteins in adult and elderly digestive conditions. Cooked meat was subjected to in vitro digestion in the dynamic multi-compartmental TIM (TNO gastroIntestinal Model) system. Digestibility and bioaccessibility were determined using nitrogen balance and digestion products were identified using mass spectrometry. The TIM model was adapted according to in vivo data to mimic the specific digestive conditions of elderly people. Meat protein digestibility and bioaccessibility were around 96 and 60% respectively and were not influenced by age (P > 0.05). As much as 800 peptides were identified in the duodenal and jejunal compartments issued from 50 meat proteins with a percentage of coverage varying from 13 to 69%. Six proteins, mainly from the cytosol, were differentially hydrolyzed under the adult and elderly digestive conditions. Pyruvate kinase was the only protein clearly showing a delay in its degradation under elderly digestive conditions. This study provides significant insights into the understanding of meat protein dynamic digestion. Such data will be helpful to design in vivo studies aiming to evaluate dietary strategies that can attenuate muscle mass loss and more generally maintain a better quality of life in the elderly population.

Comparative differential gene expression analysis of nucleus-encoded proteins for Rafflesia cantleyi against Arabidopsis thaliana

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Ng, Siuk-Mun; Lee, Xin-Wei; Wan, Kiew-Lian; Firdaus-Raih, Mohd

2015-09-01

Regulation of functional nucleus-encoded proteins targeting the plastidial functions was comparatively studied for a plant parasite, Rafflesia cantleyi versus a photosynthetic plant, Arabidopsis thaliana. This study involved two species of different feeding modes and different developmental stages. A total of 30 nucleus-encoded proteins were found to be differentially-regulated during two stages in the parasite; whereas 17 nucleus-encoded proteins were differentially-expressed during two developmental stages in Arabidopsis thaliana. One notable finding observed for the two plants was the identification of genes involved in the regulation of photosynthesis-related processes where these processes, as expected, seem to be present only in the autotroph.

A comparison of techniques for calculating protein essential dynamics

NARCIS (Netherlands)

van Aalten, D.M.F.; de Groot, B.L.; Findlay, J.B.C.; Berendsen, H.J.C.; Amadei, A

1997-01-01

Recently the basic theory of essential dynamics, a method for extracting large concerted motions from protein molecular dynamics trajectories, was described. Here, we introduce and test new aspects. A method for diagonalizing large covariance matrices is presented. We show that it is possible to

GPI-anchored protein organization and dynamics at the cell surface.

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Saha, Suvrajit; Anilkumar, Anupama Ambika; Mayor, Satyajit

2016-02-01

The surface of eukaryotic cells is a multi-component fluid bilayer in which glycosylphosphatidylinositol (GPI)-anchored proteins are an abundant constituent. In this review, we discuss the complex nature of the organization and dynamics of GPI-anchored proteins at multiple spatial and temporal scales. Different biophysical techniques have been utilized for understanding this organization, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, single particle tracking, and a number of super resolution methods. Major insights into the organization and dynamics have also come from exploring the short-range interactions of GPI-anchored proteins by fluorescence (or Förster) resonance energy transfer microscopy. Based on the nanometer to micron scale organization, at the microsecond to the second time scale dynamics, a picture of the membrane bilayer emerges where the lipid bilayer appears inextricably intertwined with the underlying dynamic cytoskeleton. These observations have prompted a revision of the current models of plasma membrane organization, and suggest an active actin-membrane composite. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Human papillomavirus E5 oncoproteins bind the A4 endoplasmic reticulum protein to regulate proliferative ability upon differentiation

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Kotnik Halavaty, Katarina; Regan, Jennifer; Mehta, Kavi; Laimins, Laimonis, E-mail: l-laimins@northwestern.edu

2014-03-15

Human papillomaviruses (HPV) infect stratified epithelia and link their life cycles to epithelial differentiation. The HPV E5 protein plays a role in the productive phase of the HPV life cycle but its mechanism of action is still unclear. We identify a new binding partner of E5, A4, using a membrane-associated yeast-two hybrid system. The A4 protein co-localizes with HPV 31 E5 in perinuclear regions and forms complexes with E5 and Bap31. In normal keratinocytes, A4 is found primarily in basal cells while in HPV positive cells high levels of A4 are seen in both undifferentiated and differentiated cells. Reduction of A4 expression by shRNAs, enhanced HPV genome amplification and increased cell proliferation ability following differentiation but this was not seen in cells lacking E5. Our studies suggest that the A4 protein is an important E5 binding partner that plays a role in regulating cell proliferation ability upon differentiation. - Highlights: • A4 associates with HPV 31 E5 proteins. • A4 is localized to endoplasmic reticulum. • HPV proteins induce A4 expression in suprabasal layers of stratified epithelium. • E5 is important for proliferation ability of differentiating HPV positive cells.

Contrasting the excited-state dynamics of the photoactive yellow protein chromophore: Protein versus solvent environments

NARCIS (Netherlands)

Vengris, M.; Horst, M.A.; Zgrablic, G.; van Stokkum, I.H.M.; Haacke, S.; Chergui, M.; Hellingwerf, K.J.; van Grondelle, R.; Larsen, D.S.

2004-01-01

Wavelength- and time-resolved fluorescence experiments have been performed on the photoactive yellow protein, the E46Q mutant, the hybrids of these proteins containing a nonisomerizing "locked" chromophore, and the native and locked chromophores in aqueous solution. The ultrafast dynamics of these

Surface N-glycoproteome patterns reveal key proteins of neuronal differentiation

Czech Academy of Sciences Publication Activity Database

Tylečková, Jiřina; Valeková, Ivona; Žižková, Martina; Rákocyová, Michaela; Maršala, S.; Maršala, M.; Gadher, S. J.; Kovářová, Hana

2016-01-01

Roč. 132, č. 1 (2016), s. 13-20 ISSN 1874-3919 R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR(CZ) TA01011466 Institutional support: RVO:67985904 Keywords : cell adhesion proteins * cell surface capture * neuronal differentiation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.914, year: 2016

Investigating the Role of Large-Scale Domain Dynamics in Protein-Protein Interactions.

Science.gov (United States)

Delaforge, Elise; Milles, Sigrid; Huang, Jie-Rong; Bouvier, Denis; Jensen, Malene Ringkjøbing; Sattler, Michael; Hart, Darren J; Blackledge, Martin

2016-01-01

Intrinsically disordered linkers provide multi-domain proteins with degrees of conformational freedom that are often essential for function. These highly dynamic assemblies represent a significant fraction of all proteomes, and deciphering the physical basis of their interactions represents a considerable challenge. Here we describe the difficulties associated with mapping the large-scale domain dynamics and describe two recent examples where solution state methods, in particular NMR spectroscopy, are used to investigate conformational exchange on very different timescales.

G-protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes.

Science.gov (United States)

O'Neill, Patrick R; Karunarathne, W K Ajith; Kalyanaraman, Vani; Silvius, John R; Gautam, N

2012-12-18

Activation of G-protein heterotrimers by receptors at the plasma membrane stimulates βγ-complex dissociation from the α-subunit and translocation to internal membranes. This intermembrane movement of lipid-modified proteins is a fundamental but poorly understood feature of cell signaling. The differential translocation of G-protein βγ-subunit types provides a valuable experimental model to examine the movement of signaling proteins between membranes in a living cell. We used live cell imaging, mathematical modeling, and in vitro measurements of lipidated fluorescent peptide dissociation from vesicles to determine the mechanistic basis of the intermembrane movement and identify the interactions responsible for differential translocation kinetics in this family of evolutionarily conserved proteins. We found that the reversible translocation is mediated by the limited affinity of the βγ-subunits for membranes. The differential kinetics of the βγ-subunit types are determined by variations among a set of basic and hydrophobic residues in the γ-subunit types. G-protein signaling thus leverages the wide variation in membrane dissociation rates among different γ-subunit types to differentially control βγ-translocation kinetics in response to receptor activation. The conservation of primary structures of γ-subunits across mammalian species suggests that there can be evolutionary selection for primary structures that confer specific membrane-binding affinities and consequent rates of intermembrane movement.

Molecular modeling of the conformational dynamics of the cellular prion protein

Science.gov (United States)

Nguyen, Charles; Colling, Ian; Bartz, Jason; Soto, Patricia

2014-03-01

Prions are infectious agents responsible for transmissible spongiform encephalopathies (TSEs), a type of fatal neurodegenerative disease in mammals. Prions propagate biological information by conversion of the non-pathological version of the prion protein to the infectious conformation, PrPSc. A wealth of knowledge has shed light on the nature and mechanism of prion protein conversion. In spite of the significance of this problem, we are far from fully understanding the conformational dynamics of the cellular isoform. To remedy this situation we employ multiple biomolecular modeling techniques such as docking and molecular dynamics simulations to map the free energy landscape and determine what specific regions of the prion protein are most conductive to binding. The overall goal is to characterize the conformational dynamics of the cell form of the prion protein, PrPc, to gain insight into inhibition pathways against misfolding. NE EPSCoR FIRST Award to Patricia Soto.

A Force Balanced Fragmentation Method for ab Initio Molecular Dynamic Simulation of Protein

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Mingyuan Xu

2018-05-01

Full Text Available A force balanced generalized molecular fractionation with conjugate caps (FB-GMFCC method is proposed for ab initio molecular dynamic simulation of proteins. In this approach, the energy of the protein is computed by a linear combination of the QM energies of individual residues and molecular fragments that account for the two-body interaction of hydrogen bond between backbone peptides. The atomic forces on the caped H atoms were corrected to conserve the total force of the protein. Using this approach, ab initio molecular dynamic simulation of an Ace-(ALA9-NME linear peptide showed the conservation of the total energy of the system throughout the simulation. Further a more robust 110 ps ab initio molecular dynamic simulation was performed for a protein with 56 residues and 862 atoms in explicit water. Compared with the classical force field, the ab initio molecular dynamic simulations gave better description of the geometry of peptide bonds. Although further development is still needed, the current approach is highly efficient, trivially parallel, and can be applied to ab initio molecular dynamic simulation study of large proteins.

Tokamak turbulence in self-regulated differentially rotating flow and L-H transition dynamics

International Nuclear Information System (INIS)

Terry, P.W.; Carreras, B.A.; Sidikman, K.

1992-01-01

An analytical study of turbulence in the presence of turbulently generated differentially rotating flow is presented as a paradigm for fluctuation dynamics in L- and H-mode plasmas. Using a drift wave model, the role of both flow shear and flow curvature (second radial derivative of the poloidal ExB flow) is detailed in linear and saturated turbulence phases. In the strong turbulence saturated state, finite amplitude-induced modification of the fluctuation structure near low order rational surfaces strongly inhibits flow shear suppression. Suppression by curvature is not diminished, but it occurs through a frequency shift. A description of L-H mode transition dynamics based on the self-consistent linking of turbulence suppression by differentially rotating flow and generation of flow by turbulent momentum transport is presented. In this model, rising edge temperature triggers a transition characterized by spontaneous generation of differentially rotating flow and decreasing turbulence intensity

New technique of identifying the hierarchy of dynamic domains in proteins using a method of molecular dynamics simulations

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Yesylevskyy S. O.

2010-04-01

Full Text Available Aim. Despite a large number of existing domain identification techniques there is no universally accepted method, which identifies the hierarchy of dynamic domains using the data of molecular dynamics (MD simulations. The goal of this work is to develop such technique. Methods. The dynamic domains are identified by eliminating systematic motions from MD trajectories recursively in a model-free manner. Results. The technique called the Hierarchical Domain-Wise Alignment (HDWA to identify hierarchically organized dynamic domains in proteins using the MD trajectories has been developed. Conclusion. A new method of domain identification in proteins is proposed

The diagnostic value of c-reactive protein estimation in differentiating bacterial from viral meningitis

International Nuclear Information System (INIS)

Sheikh, A.

2001-01-01

Objective: To evaluate the efficacy of serum and CSF C-reactive protein (C-rp) in differentiating bacterial from viral meningitis. Design: An observational, respective hospital-based study. Place and duration of study: It was conducted at the Department of Medicine and Department of Pediatrics, Shaikh Zayed Postgraduate Medical Institute Lahore, Over a Period of one year between march, 1999 and March, 2000. Subject and Methods: A randomized group of thirty patients, who presented with clinical features, suggestive of meningitis, were included in the study. C-reactive protein determinations were performed by latex agglutination method on the serum and cerebrospinal fluid (CSF) of these patients. Results: In the present study, c-reactive protein was found to be a more sensitive test for differentiating bacterial from non-bacterial meningitis on initial examination than the usual conventional methods used to diagnose bacterial meningitis. CSF C-reactive protein had a greater sensitivity (92% as compared to serum C-reactive protein (71%). Conclusion: C-reactive protein determination in CSF was found to be a useful indicator of bacterial meningitis that can be used to distinguish it from viral meningitis. (author)

Protein profiling in potato (Solanum tuberosum L.) leaf tissues by differential centrifugation.

Science.gov (United States)

Lim, Sanghyun; Chisholm, Kenneth; Coffin, Robert H; Peters, Rick D; Al-Mughrabi, Khalil I; Wang-Pruski, Gefu; Pinto, Devanand M

2012-04-06

Foliar diseases, such as late blight, result in serious threats to potato production. As such, potato leaf tissue becomes an important substrate to study biological processes, such as plant defense responses to infection. Nonetheless, the potato leaf proteome remains poorly characterized. Here, we report protein profiling of potato leaf tissues using a modified differential centrifugation approach to separate the leaf tissues into cell wall and cytoplasmic fractions. This method helps to increase the number of identified proteins, including targeted putative cell wall proteins. The method allowed for the identification of 1484 nonredundant potato leaf proteins, of which 364 and 447 were reproducibly identified proteins in the cell wall and cytoplasmic fractions, respectively. Reproducibly identified proteins corresponded to over 70% of proteins identified in each replicate. A diverse range of proteins was identified based on their theoretical pI values, molecular masses, functional classification, and biological processes. Such a protein extraction method is effective for the establishment of a highly qualified proteome profile.

Analysis of differentially expressed proteins in Yersinia enterocolitica-infected HeLa cells.

Science.gov (United States)

Alugubelly, Navatha; Hercik, Kamil; Kibler, Peter; Nanduri, Bindu; Edelmann, Mariola J

2016-05-01

Yersinia enterocolitica is a facultative intracellular pathogen and a causative agent of yersiniosis, which can be contracted by ingestion of contaminated food. Yersinia secretes virulence factors to subvert critical pathways in the host cell. In this study we utilized shotgun label-free proteomics to study differential protein expression in epithelial cells infected with Y.enterocolitica. We identified a total of 551 proteins, amongst which 42 were downregulated (including Prostaglandin E Synthase 3, POH-1 and Karyopherin alpha) and 22 were upregulated (including Rab1 and RhoA) in infected cells. We validated some of these results by western blot analysis of proteins extracted from Caco-2 and HeLa cells. The proteomic dataset was used to identify host canonical pathways and molecular functions modulated by this infection in the host cells. This study constitutes a proteome of Yersinia-infected cells and can support new discoveries in the area of host-pathogen interactions. We describe a proteome of Yersinia enterocolitica-infected HeLa cells, including a description of specific proteins differentially expressed upon infection, molecular functions as well as pathways altered during infection. This proteomic study can lead to a better understanding of Y. enterocolitica pathogenesis in human epithelial cells. Copyright © 2016. Published by Elsevier B.V.

Differential Expression of Proteins Associated with the Hair Follicle Cycle - Proteomics and Bioinformatics Analyses.

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Lei Wang

Full Text Available Hair follicle cycling can be divided into the following three stages: anagen, catagen, and telogen. The molecular signals that orchestrate the follicular transition between phases are still unknown. To better understand the detailed protein networks controlling this process, proteomics and bioinformatics analyses were performed to construct comparative protein profiles of mouse skin at specific time points (0, 8, and 20 days. Ninety-five differentially expressed protein spots were identified by MALDI-TOF/TOF as 44 proteins, which were found to change during hair follicle cycle transition. Proteomics analysis revealed that these changes in protein expression are involved in Ca2+-regulated biological processes, migration, and regulation of signal transduction, among other processes. Subsequently, three proteins were selected to validate the reliability of expression patterns using western blotting. Cluster analysis revealed three expression patterns, and each pattern correlated with specific cell processes that occur during the hair cycle. Furthermore, bioinformatics analysis indicated that the differentially expressed proteins impacted multiple biological networks, after which detailed functional analyses were performed. Taken together, the above data may provide insight into the three stages of mouse hair follicle morphogenesis and provide a solid basis for potential therapeutic molecular targets for this hair disease.

Investigating the Role of Large-Scale Domain Dynamics in Protein-Protein Interactions

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Elise Delaforge

2016-09-01

Full Text Available Intrinsically disordered linkers provide multi-domain proteins with degrees of conformational freedom that are often essential for function. These highly dynamic assemblies represent a significant fraction of all proteomes, and deciphering the physical basis of their interactions represents a considerable challenge. Here we describe the difficulties associated with mapping the large-scale domain dynamics and describe two recent examples where solution state methods, in particular NMR spectroscopy, are used to investigate conformational exchange on very different timescales.

Dynamics of the association of heat shock protein HSPA6 (Hsp70B') and HSPA1A (Hsp70-1) with stress-sensitive cytoplasmic and nuclear structures in differentiated human neuronal cells.

Science.gov (United States)

Shorbagi, Sadek; Brown, Ian R

2016-11-01

Heat shock proteins (Hsps) are cellular repair agents that counter the effects of protein misfolding that is a characteristic feature of neurodegenerative diseases. HSPA1A (Hsp70-1) is a widely studied member of the HSPA (Hsp70) family. The little-studied HSPA6 (Hsp70B') is present in the human genome and absent in mouse and rat; hence, it is missing in current animal models of neurodegenerative diseases. Differentiated human neuronal SH-SY5Y cells were employed to compare the dynamics of the association of YFP-tagged HSPA6 and HSPA1A with stress-sensitive cytoplasmic and nuclear structures. Following thermal stress, live-imaging confocal microscopy and Fluorescence Recovery After Photobleaching (FRAP) demonstrated that HSPA6 displayed a prolonged and more dynamic association, compared to HSPA1A, with centrioles that play critical roles in neuronal polarity and migration. HSPA6 and HSPA1A also targeted nuclear speckles, rich in RNA splicing factors, and the granular component of the nucleolus that is involved in rRNA processing and ribosomal subunit assembly. HSPA6 and HSPA1A displayed similar FRAP kinetics in their interaction with nuclear speckles and the nucleolus. Subsequently, during the recovery from neuronal stress, HSPA6, but not HSPA1A, localized with the periphery of nuclear speckles (perispeckles) that have been characterized as transcription sites. The stress-induced association of HSPA6 with perispeckles displayed the greatest dynamism compared to the interaction of HSPA6 or HSPA1A with other stress-sensitive cytoplasmic and nuclear structures. This suggests involvement of HSPA6 in transcriptional recovery of human neurons from cellular stress that is not apparent for HSPA1A.

Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes

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He, Yonghan [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China); Li, Ying [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Zhang, Shuocheng [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Perry, Ben [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Zhao, Tiantian [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Psychology, University of Toronto, 1265 Military Trail, Toronto, ON, Canada M1C 1A4 (Canada); Wang, Yanwen, E-mail: yanwen.wang@nrc.ca [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Sun, Changhao, E-mail: sun2002changhao@yahoo.com [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China)

2013-06-28

Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR{sub γ}) and CCAAT element binding protein α (C/EBP{sub α}), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.

Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes

International Nuclear Information System (INIS)

He, Yonghan; Li, Ying; Zhang, Shuocheng; Perry, Ben; Zhao, Tiantian; Wang, Yanwen; Sun, Changhao

2013-01-01

Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR γ ) and CCAAT element binding protein α (C/EBP α ), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins

Differential protein modulation in midguts of Aedes aegypti infected with chikungunya and dengue 2 viruses.

Directory of Open Access Journals (Sweden)

Stéphane Tchankouo-Nguetcheu

Full Text Available BACKGROUND: Arthropod borne virus infections cause several emerging and resurgent infectious diseases. Among the diseases caused by arboviruses, dengue and chikungunya are responsible for a high rate of severe human diseases worldwide. The midgut of mosquitoes is the first barrier for pathogen transmission and is a target organ where arboviruses must replicate prior to infecting other organs. A proteomic approach was undertaken to characterize the key virus/vector interactions and host protein modifications that happen in the midgut for viral transmission to eventually take place. METHODOLOGY AND PRINCIPAL FINDINGS: Using a proteomics differential approach with two-Dimensional Differential in-Gel Electrophoresis (2D-DIGE, we defined the protein modulations in the midgut of Aedes aegypti that were triggered seven days after an oral infection (7 DPI with dengue 2 (DENV-2 and chikungunya (CHIKV viruses. Gel profile comparisons showed that the level of 18 proteins was modulated by DENV-2 only and 12 proteins were modulated by CHIKV only. Twenty proteins were regulated by both viruses in either similar or different ways. Both viruses caused an increase of proteins involved in the generation of reactive oxygen species, energy production, and carbohydrate and lipid metabolism. Midgut infection by DENV-2 and CHIKV triggered an antioxidant response. CHIKV infection produced an increase of proteins involved in detoxification. CONCLUSION/SIGNIFICANCE: Our study constitutes the first analysis of the protein response of Aedes aegypti's midgut infected with viruses belonging to different families. It shows that the differentially regulated proteins in response to viral infection include structural, redox, regulatory proteins, and enzymes for several metabolic pathways. Some of these proteins like antioxidant are probably involved in cell protection. On the other hand, we propose that the modulation of other proteins like transferrin, hsp60 and alpha

Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells.

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Sugiyama, Hayami; Takahashi, Kazutoshi; Yamamoto, Takuya; Iwasaki, Mio; Narita, Megumi; Nakamura, Masahiro; Rand, Tim A; Nakagawa, Masato; Watanabe, Akira; Yamanaka, Shinya

2017-01-10

Novel APOBEC1 target 1 (Nat1) (also known as "p97," "Dap5," and "Eif4g2") is a ubiquitously expressed cytoplasmic protein that is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (Eif4g1). We previously showed that Nat1-null mouse embryonic stem cells (mES cells) are resistant to differentiation. In the current study, we found that NAT1 and eIF4G1 share many binding proteins, such as the eukaryotic translation initiation factors eIF3 and eIF4A and ribosomal proteins. However, NAT1 did not bind to eIF4E or poly(A)-binding proteins, which are critical for cap-dependent translation initiation. In contrast, compared with eIF4G1, NAT1 preferentially interacted with eIF2, fragile X mental retardation proteins (FMR), and related proteins and especially with members of the proline-rich and coiled-coil-containing protein 2 (PRRC2) family. We also found that Nat1-null mES cells possess a transcriptional profile similar, although not identical, to the ground state, which is established in wild-type mES cells when treated with inhibitors of the ERK and glycogen synthase kinase 3 (GSK3) signaling pathways. In Nat1-null mES cells, the ERK pathway is suppressed even without inhibitors. Ribosome profiling revealed that translation of mitogen-activated protein kinase kinase kinase 3 (Map3k3) and son of sevenless homolog 1 (Sos1) is suppressed in the absence of Nat1 Forced expression of Map3k3 induced differentiation of Nat1-null mES cells. These data collectively show that Nat1 is involved in the translation of proteins that are required for cell differentiation.

Dynamic gastric digestion of a commercial whey protein concentrate†.

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Miralles, Beatriz; Del Barrio, Roberto; Cueva, Carolina; Recio, Isidra; Amigo, Lourdes

2018-03-01

A dynamic gastrointestinal simulator, simgi ® , has been applied to assess the gastric digestion of a whey protein concentrate. Samples collected from the outlet of the stomach have been compared to those resulting from the static digestion protocol INFOGEST developed on the basis of physiologically inferred conditions. Progress of digestion was followed by SDS-PAGE and LC-MS/MS. By SDS-PAGE, serum albumin and α-lactalbumin were no longer detectable at 30 and 60 min, respectively. On the contrary, β-lactoglobulin was visible up to 120 min, although in decreasing concentrations in the dynamic model due to the gastric emptying and the addition of gastric fluids. Moreover, β-lactoglobulin was partly hydrolysed by pepsin probably due to the presence of heat-denatured forms and the peptides released using both digestion models were similar. Under dynamic conditions, a stepwise increase in number of peptides over time was observed, while the static protocol generated a high number of peptides from the beginning of digestion. Whey protein digestion products using a dynamic stomach are consistent with those generated with the static protocol but the kinetic behaviour of the peptide profile emphasises the effect of the sequential pepsin addition, peristaltic shaking, and gastric emptying on protein digestibility. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Applications of differential algebra to single-particle dynamics in storage rings

International Nuclear Information System (INIS)

Yan, Y.

1991-09-01

Recent developments in the use of differential algebra to study single-particle beam dynamics in charged-particle storage rings are the subject of this paper. Chapter 2 gives a brief review of storage rings. The concepts of betatron motion and synchrotron motion, and their associated resonances, are introduced. Also introduced are the concepts of imperfections, such as off-momentum, misalignment, and random and systematic errors, and their associated corrections. The chapter concludes with a discussion of numerical simulation principles and the concept of one-turn periodic maps. In Chapter 3, the discussion becomes more focused with the introduction of differential algebras. The most critical test for differential algebraic mapping techniques -- their application to long-term stability studies -- is discussed in Chapter 4. Chapter 5 presents a discussion of differential algebraic treatment of dispersed betatron motion. The paper concludes in Chapter 6 with a discussion of parameterization of high-order maps

Collective dynamics of protein hydration water by brillouin neutron spectroscopy.

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Orecchini, Andrea; Paciaroni, Alessandro; De Francesco, Alessio; Petrillo, Caterina; Sacchetti, Francesco

2009-04-08

By a detailed experimental study of THz dynamics in the ribonuclease protein, we could detect the propagation of coherent collective density fluctuations within the protein hydration shell. The emerging picture indicates the presence of both a dispersing mode, traveling with a speed greater than 3000 m/s, and a nondispersing one, characterized by an almost constant energy of 6-7 meV. In agreement with molecular dynamics simulations [Phys. Rev. Lett. 2002, 89, 275501], the features of the dispersion curves closely resemble those observed in pure liquid water [Phys. Rev. E: Stat. Phys., Plasmas, Fluids, Relat. Interdiscip. Top. 2004, 69, 061203]. On the contrary, the observed damping factors are much larger than in bulk water, with the dispersing mode becoming overdamped at Q = 0.6 A(-1) already. Such novel experimental findings are discussed as a dynamic signature of the disordering effect induced by the protein surface on the local structure of water.

Protein Malnutrition Induces Bone Marrow Mesenchymal Stem Cells Commitment to Adipogenic Differentiation Leading to Hematopoietic Failure

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Cunha, Mayara Caldas Ramos; Lima, Fabiana da Silva; Vinolo, Marco Aurélio Ramirez; Hastreiter, Araceli; Curi, Rui; Borelli, Primavera; Fock, Ricardo Ambrósio

2013-01-01

Protein malnutrition (PM) results in pathological changes that are associated with peripheral leukopenia, bone marrow (BM) hypoplasia and alterations in the BM microenvironment leading to hematopoietic failure; however, the mechanisms involved are poorly understood. In this context, the BM mesenchymal stem cells (MSCs) are cells intimately related to the formation of the BM microenvironment, and their differentiation into adipocytes is important because adipocytes are cells that have the capability to negatively modulate hematopoiesis. Two-month-old male Balb/c mice were subjected to protein-energy malnutrition with a low-protein diet containing 2% protein, whereas control animals were fed a diet containing 12% protein. The hematopoietic parameters and the expression of CD45 and CD117 positive cells in the BM were evaluated. MSCs were isolated from BM, and their capability to produce SCF, IL-3, G-CSF and GM-CSF were analyzed. The expression of PPAR-γ and C/EBP-α as well as the expression of PPAR-γ and SREBP mRNAs were evaluated in MSCs together with their capability to differentiate into adipocytes in vitro. The malnourished animals had anemia and leukopenia as well as spleen and bone marrow hypoplasia and a reduction in the expression of CD45 and CD117 positive cells from BM. The MSCs of the malnourished mice presented an increased capability to produce SCF and reduced production of G-CSF and GM-CSF. The MSCs from the malnourished animals showed increased expression of PPAR-γ protein and PPAR-γ mRNA associated with an increased capability to differentiate into adipocytes. The alterations found in the malnourished animals allowed us to conclude that malnutrition committed MSC differentiation leading to adipocyte decision and compromised their capacity for cytokine production, contributing to an impaired hematopoietic microenvironment and inducing the bone marrow failure commonly observed in protein malnutrition states. PMID:23516566

Protein malnutrition induces bone marrow mesenchymal stem cells commitment to adipogenic differentiation leading to hematopoietic failure.

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Cunha, Mayara Caldas Ramos; Lima, Fabiana da Silva; Vinolo, Marco Aurélio Ramirez; Hastreiter, Araceli; Curi, Rui; Borelli, Primavera; Fock, Ricardo Ambrósio

2013-01-01

Protein malnutrition (PM) results in pathological changes that are associated with peripheral leukopenia, bone marrow (BM) hypoplasia and alterations in the BM microenvironment leading to hematopoietic failure; however, the mechanisms involved are poorly understood. In this context, the BM mesenchymal stem cells (MSCs) are cells intimately related to the formation of the BM microenvironment, and their differentiation into adipocytes is important because adipocytes are cells that have the capability to negatively modulate hematopoiesis. Two-month-old male Balb/c mice were subjected to protein-energy malnutrition with a low-protein diet containing 2% protein, whereas control animals were fed a diet containing 12% protein. The hematopoietic parameters and the expression of CD45 and CD117 positive cells in the BM were evaluated. MSCs were isolated from BM, and their capability to produce SCF, IL-3, G-CSF and GM-CSF were analyzed. The expression of PPAR-γ and C/EBP-α as well as the expression of PPAR-γ and SREBP mRNAs were evaluated in MSCs together with their capability to differentiate into adipocytes in vitro. The malnourished animals had anemia and leukopenia as well as spleen and bone marrow hypoplasia and a reduction in the expression of CD45 and CD117 positive cells from BM. The MSCs of the malnourished mice presented an increased capability to produce SCF and reduced production of G-CSF and GM-CSF. The MSCs from the malnourished animals showed increased expression of PPAR-γ protein and PPAR-γ mRNA associated with an increased capability to differentiate into adipocytes. The alterations found in the malnourished animals allowed us to conclude that malnutrition committed MSC differentiation leading to adipocyte decision and compromised their capacity for cytokine production, contributing to an impaired hematopoietic microenvironment and inducing the bone marrow failure commonly observed in protein malnutrition states.

bHLH-O proteins balance the self-renewal and differentiation of Drosophila neural stem cells by regulating Earmuff expression.

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Li, Xiaosu; Chen, Rui; Zhu, Sijun

2017-11-15

Balancing self-renewal and differentiation of stem cells requires differential expression of self-renewing factors in two daughter cells generated from the asymmetric division of the stem cells. In Drosophila type II neural stem cell (or neuroblast, NB) lineages, the expression of the basic helix-loop-helix-Orange (bHLH-O) family proteins, including Deadpan (Dpn) and E(spl) proteins, is required for maintaining the self-renewal and identity of type II NBs, whereas the absence of these self-renewing factors is essential for the differentiation of intermediate neural progenitors (INPs) generated from type II NBs. Here, we demonstrate that Dpn maintains type II NBs by suppressing the expression of Earmuff (Erm). We provide evidence that Dpn and E(spl) proteins suppress Erm by directly binding to C-sites and N-boxes in the cis-regulatory region of erm. Conversely, the absence of bHLH-O proteins in INPs allows activation of erm and Erm-mediated maturation of INPs. Our results further suggest that Pointed P1 (PntP1) mediates the dedifferentiation of INPs resulting from the loss of Erm or overexpression of Dpn or E(spl) proteins. Taken together, these findings reveal mechanisms underlying the regulation of the maintenance of type II NBs and differentiation of INPs through the differential expression of bHLH-O family proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Efficient myogenic differentiation of human adipose-derived stem cells by the transduction of engineered MyoD protein

Energy Technology Data Exchange (ETDEWEB)

Sung, Min Sun [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350 (Korea, Republic of); Mun, Ji-Young [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Kwon, Ohsuk [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350 (Korea, Republic of); Kwon, Ki-Sun [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Oh, Doo-Byoung, E-mail: dboh@kribb.re.kr [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350 (Korea, Republic of)

2013-07-19

Highlights: •MyoD was engineered to contain protein transduction domain and endosome-disruptive INF7 peptide. •The engineered MyoD-IT showed efficient nuclear targeting through an endosomal escape by INF7 peptide. •By applying MyoD-IT, human adipose-derived stem cells (hASCs) were differentiated into myogenic cells. •hASCs differentiated by applying MyoD-IT fused to myotubes through co-culturing with mouse myoblasts. •Myogenic differentiation using MyoD-IT is a safe method without the concern of altering the genome. -- Abstract: Human adipose-derived stem cells (hASCs) have great potential as cell sources for the treatment of muscle disorders. To provide a safe method for the myogenic differentiation of hASCs, we engineered the MyoD protein, a key transcription factor for myogenesis. The engineered MyoD (MyoD-IT) was designed to contain the TAT protein transduction domain for cell penetration and the membrane-disrupting INF7 peptide, which is an improved version of the HA2 peptide derived from influenza. MyoD-IT showed greatly improved nuclear targeting ability through an efficient endosomal escape induced by the pH-sensitive membrane disruption of the INF7 peptide. By applying MyoD-IT to a culture, hASCs were efficiently differentiated into long spindle-shaped myogenic cells expressing myosin heavy chains. Moreover, these cells differentiated by an application of MyoD-IT fused to myotubes with high efficiency through co-culturing with mouse C2C12 myoblasts. Because internalized proteins can be degraded in cells without altering the genome, the myogenic differentiation of hASCs using MyoD-IT would be a safe and clinically applicable method.

Efficient myogenic differentiation of human adipose-derived stem cells by the transduction of engineered MyoD protein

International Nuclear Information System (INIS)

Sung, Min Sun; Mun, Ji-Young; Kwon, Ohsuk; Kwon, Ki-Sun; Oh, Doo-Byoung

2013-01-01

Highlights: •MyoD was engineered to contain protein transduction domain and endosome-disruptive INF7 peptide. •The engineered MyoD-IT showed efficient nuclear targeting through an endosomal escape by INF7 peptide. •By applying MyoD-IT, human adipose-derived stem cells (hASCs) were differentiated into myogenic cells. •hASCs differentiated by applying MyoD-IT fused to myotubes through co-culturing with mouse myoblasts. •Myogenic differentiation using MyoD-IT is a safe method without the concern of altering the genome. -- Abstract: Human adipose-derived stem cells (hASCs) have great potential as cell sources for the treatment of muscle disorders. To provide a safe method for the myogenic differentiation of hASCs, we engineered the MyoD protein, a key transcription factor for myogenesis. The engineered MyoD (MyoD-IT) was designed to contain the TAT protein transduction domain for cell penetration and the membrane-disrupting INF7 peptide, which is an improved version of the HA2 peptide derived from influenza. MyoD-IT showed greatly improved nuclear targeting ability through an efficient endosomal escape induced by the pH-sensitive membrane disruption of the INF7 peptide. By applying MyoD-IT to a culture, hASCs were efficiently differentiated into long spindle-shaped myogenic cells expressing myosin heavy chains. Moreover, these cells differentiated by an application of MyoD-IT fused to myotubes with high efficiency through co-culturing with mouse C2C12 myoblasts. Because internalized proteins can be degraded in cells without altering the genome, the myogenic differentiation of hASCs using MyoD-IT would be a safe and clinically applicable method

Secreted Frizzled related protein-4 (sFRP4) promotes epidermal differentiation and apoptosis

International Nuclear Information System (INIS)

Maganga, Richard; Giles, Natalie; Adcroft, Katharine; Unni, Ambili; Keeney, Diane; Wood, Fiona; Fear, Mark; Dharmarajan, Arunasalam

2008-01-01

The skin provides vital protection from infection and dehydration. Maintenance of the skin is through a constant program of proliferation, differentiation and apoptosis of epidermal cells, whereby proliferating cells in the basal layer differentiating to form the keratinized, anucleated stratum corneum. The WNT signalling pathway is known to be important in the skin. WNT signalling has been shown to be important both in epidermal development and in the maintenance and cycling of hair follicles and epidermal stem cells. However, the precise role for this pathway in epidermal differentiation remains unknown. We investigated the role of the WNT signalling inhibitor sFRP4 in epidermal differentiation. sFRP4 is expressed in both normal skin and keratinocytes in culture. Expression of sFRP4 mRNA and protein increases with keratinocyte differentiation and apoptosis, whilst exposure of keratinocytes to exogenous sFRP4 promotes apoptosis and expression of the terminal differentiation marker Involucrin. These data suggest sFRP4 promotes epidermal differentiation.

Dynamics differentiate between active and inactive inteins.

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Cronin, Melissa; Coolbaugh, Michael J; Nellis, David; Zhu, Jianwei; Wood, David W; Nussinov, Ruth; Ma, Buyong

2015-02-16

The balance between stability and dynamics for active enzymes can be somewhat quantified by studies of intein splicing and cleaving reactions. Inteins catalyze the ligation of flanking host exteins while excising themselves. The potential for applications led to engineering of a mini-intein splicing domain, where the homing endonuclease domain of the Mycobacterium tuberculosis RecA (Mtu recA) intein was removed. The remaining domains were linked by several short peptides, but splicing activity in all was substantially lower than the full-length intein. Native splicing activity was restored in some cases by a V67L mutation. Using computations and experiments, we examine the impact of this mutation on the stability and conformational dynamics of the mini-intein splicing domain. Molecular dynamics simulations were used to delineate the factors that determine the active state, including the V67L mini-intein mutant, and peptide linker. We found that (1) the V67L mutation lowers the global fluctuations in all modeled mini-inteins, stabilizing the mini-intein constructs; (2) the connecting linker length affects intein dynamics; and (3) the flexibilities of the linker and intein core are higher in the active structure. We have observed that the interaction of the linker region and a turn region around residues 35-41 provides the pathway for the allostery interaction. Our experiments reveal that intein catalysis is characterized by non-linear Arrhenius plot, confirming the significant contribution of protein conformational dynamics to intein function. We conclude that while the V67L mutation stabilizes the global structure, cooperative dynamics of all intein regions appear more important for intein function than high stability. Our studies suggest that effectively quenching the conformational dynamics of an intein through engineered allosteric interactions could deactivate intein splicing or cleaving. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Dynamic and bio-orthogonal protein assembly along a supramolecular polymer

NARCIS (Netherlands)

Petkau - Milroy, K.; Uhlenheuer, D.A.; Spiering, A.J.H.; Vekemans, J.A.J.M.; Brunsveld, L.

2013-01-01

Dynamic protein assembly along supramolecular columnar polymers has been achieved through the site-specific covalent attachment of different SNAP-tag fusion proteins to self-assembled benzylguanine-decorated discotics. The self-assembly of monovalent discotics into supramolecular polymers creates a

In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

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Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

2010-07-01

This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination.

Directory of Open Access Journals (Sweden)

Samantha F Kornfeld

Full Text Available Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and dystonia musculorum in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of dystonia musculorum, Dstdt-27J, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of Dstdt-27J oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between Dstdt-27J and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that Dstdt-27J oligodendrocyte progenitor cells were able to migrate normally. Finally, in vivo analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of Dstdt-27J optic nerves was normal, as

Delay differential systems for tick population dynamics.

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Fan, Guihong; Thieme, Horst R; Zhu, Huaiping

2015-11-01

Ticks play a critical role as vectors in the transmission and spread of Lyme disease, an emerging infectious disease which can cause severe illness in humans or animals. To understand the transmission dynamics of Lyme disease and other tick-borne diseases, it is necessary to investigate the population dynamics of ticks. Here, we formulate a system of delay differential equations which models the stage structure of the tick population. Temperature can alter the length of time delays in each developmental stage, and so the time delays can vary geographically (and seasonally which we do not consider). We define the basic reproduction number [Formula: see text] of stage structured tick populations. The tick population is uniformly persistent if [Formula: see text] and dies out if [Formula: see text]. We present sufficient conditions under which the unique positive equilibrium point is globally asymptotically stable. In general, the positive equilibrium can be unstable and the system show oscillatory behavior. These oscillations are primarily due to negative feedback within the tick system, but can be enhanced by the time delays of the different developmental stages.

Identification of differentially expressed proteins between human esophageal immortalized and carcinomatous cell lines by two-dimensional electrophoresis and MALDI-TOF-mass spectrometry

Institute of Scientific and Technical Information of China (English)

Xing-Dong Xiong; Li-Yan Xu; Zhong-Ying Shen; Wei-Jia Cai; Jian-Min Luo; Ya-Li Han; En-Min Li

2002-01-01

AIM: To identify the differentially expressed proteins between the human immortalized esophageal epithelial cell line (SHEE) and the malignant transformed esophageal carcinoma cell line (SHEEC), and to explore new ways for studying esophageal carcinoma associated genes. METHODS: SHEE and SHEEC cell lines were used to separate differentially expressed proteins by two-dimensional electrophoresis/The silver-stained 2-D gels was scanned with EDAS290 digital camera system and analyzed with the PDQuest 6.2 Software. Six spots in which the differentially expressed protein was more obvious were selected and analyzed with matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS).RESULTS: There were 107±4.58 and 115±9.91 protein spots observed in SHEE and SHEEC respectively, and the majority of these spots between the two cell lines matched each other (r＝-0.772), only a few were expressed differentially. After analyzed by MALDI-TOF-MS and database search for the six differentially expressed proteins, One new protein as well as other five sequence-known proteins including RNPEP-like protein, human rRNA gene upstream sequence binding transcription factor, uracil DNA glycosylase,Annexin A2 and p300/CBP-associated factor were preliminarily identified.CONCLUSION: These differentially expressed proteins might play an importance role during malignant transformation of SHEEC from SHEE. The identification of these proteins may serve as a new way for studying esophageal carcinoma associated genes.

Dynamically polarized samples for neutron protein crystallography at the Spallation Neutron Source

International Nuclear Information System (INIS)

Zhao, Jinkui; Pierce, Josh; Robertson, J. L.; Herwig, Kenneth W.; Myles, Dean; Cuneo, Matt; Li, Le; Meilleur, Flora; Standaert, Bob

2016-01-01

To prepare for the next generation neutron scattering instruments for the planned second target station at the Spallation Neutron Source (SNS) and to broaden the scientific impact of neutron protein crystallography at the Oak Ridge National Laboratory, we have recently ramped up our efforts to develop a dynamically polarized target for neutron protein crystallography at the SNS. Proteins contain a large amount of hydrogen which contributes to incoherent diffraction background and limits the sensitivity of neutron protein crystallography. This incoherent background can be suppressed by using polarized neutron diffraction, which in the same time also improves the coherent diffraction signal. Our plan is to develop a custom Dynamic Nuclear Polarization (DNP) setup tailored to neutron protein diffraction instruments. Protein crystals will be polarized at a magnetic field of 5 T and temperatures of below 1 K. After the dynamic polarization process, the sample will be brought to a frozen-spin mode in a 0.5 T holding field and at temperatures below 100 mK. In a parallel effort, we are also investigating various ways of incorporating polarization agents needed for DNP, such as site specific spin labels, into protein crystals. (paper)

A sensitive fluorescent probe for the polar solvation dynamics at protein-surfactant interfaces.

Science.gov (United States)

Singh, Priya; Choudhury, Susobhan; Singha, Subhankar; Jun, Yongwoong; Chakraborty, Sandipan; Sengupta, Jhimli; Das, Ranjan; Ahn, Kyo-Han; Pal, Samir Kumar

2017-05-17

Relaxation dynamics at the surface of biologically important macromolecules is important taking into account their functionality in molecular recognition. Over the years it has been shown that the solvation dynamics of a fluorescent probe at biomolecular surfaces and interfaces account for the relaxation dynamics of polar residues and associated water molecules. However, the sensitivity of the dynamics depends largely on the localization and exposure of the probe. For noncovalent fluorescent probes, localization at the region of interest in addition to surface exposure is an added challenge compared to the covalently attached probes at the biological interfaces. Here we have used a synthesized donor-acceptor type dipolar fluorophore, 6-acetyl-(2-((4-hydroxycyclohexyl)(methyl)amino)naphthalene) (ACYMAN), for the investigation of the solvation dynamics of a model protein-surfactant interface. A significant structural rearrangement of a model histone protein (H1) upon interaction with anionic surfactant sodium dodecyl sulphate (SDS) as revealed from the circular dichroism (CD) studies is nicely corroborated in the solvation dynamics of the probe at the interface. The polarization gated fluorescence anisotropy of the probe compared to that at the SDS micellar surface clearly reveals the localization of the probe at the protein-surfactant interface. We have also compared the sensitivity of ACYMAN with other solvation probes including coumarin 500 (C500) and 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl)-4H-pyran (DCM). In comparison to ACYMAN, both C500 and DCM fail to probe the interfacial solvation dynamics of a model protein-surfactant interface. While C500 is found to be delocalized from the protein-surfactant interface, DCM becomes destabilized upon the formation of the interface (protein-surfactant complex). The timescales obtained from this novel probe have also been compared with other femtosecond resolved studies and molecular dynamics simulations.

TENSOR CALCULUS with applications to Differential Theory of Surfaces and Dynamics

DEFF Research Database (Denmark)

Nielsen, Søren R. K.

The present outline on tensor calculus with special application to differential theory of surfaces and dynamics represents a modified and extended version of a lecture note written by the author as an introduction to a course on shell theory given together with Ph.D. Jesper Winther Stærdahl...

Applying differential dynamic logic to reconfigurable biological networks.

Science.gov (United States)

Figueiredo, Daniel; Martins, Manuel A; Chaves, Madalena

2017-09-01

Qualitative and quantitative modeling frameworks are widely used for analysis of biological regulatory networks, the former giving a preliminary overview of the system's global dynamics and the latter providing more detailed solutions. Another approach is to model biological regulatory networks as hybrid systems, i.e., systems which can display both continuous and discrete dynamic behaviors. Actually, the development of synthetic biology has shown that this is a suitable way to think about biological systems, which can often be constructed as networks with discrete controllers, and present hybrid behaviors. In this paper we discuss this approach as a special case of the reconfigurability paradigm, well studied in Computer Science (CS). In CS there are well developed computational tools to reason about hybrid systems. We argue that it is worth applying such tools in a biological context. One interesting tool is differential dynamic logic (dL), which has recently been developed by Platzer and applied to many case-studies. In this paper we discuss some simple examples of biological regulatory networks to illustrate how dL can be used as an alternative, or also as a complement to methods already used. Copyright © 2017 Elsevier Inc. All rights reserved.

Pluripotency factors in embryonic stem cells regulate differentiation into germ layers.

Science.gov (United States)

Thomson, Matt; Liu, Siyuan John; Zou, Ling-Nan; Smith, Zack; Meissner, Alexander; Ramanathan, Sharad

2011-06-10

Cell fate decisions are fundamental for development, but we do not know how transcriptional networks reorganize during the transition from a pluripotent to a differentiated cell state. Here, we asked how mouse embryonic stem cells (ESCs) leave the pluripotent state and choose between germ layer fates. By analyzing the dynamics of the transcriptional circuit that maintains pluripotency, we found that Oct4 and Sox2, proteins that maintain ESC identity, also orchestrate germ layer fate selection. Oct4 suppresses neural ectodermal differentiation and promotes mesendodermal differentiation; Sox2 inhibits mesendodermal differentiation and promotes neural ectodermal differentiation. Differentiation signals continuously and asymmetrically modulate Oct4 and Sox2 protein levels, altering their binding pattern in the genome, and leading to cell fate choice. The same factors that maintain pluripotency thus also integrate external signals and control lineage selection. Our study provides a framework for understanding how complex transcription factor networks control cell fate decisions in progenitor cells. Copyright © 2011 Elsevier Inc. All rights reserved.

Role of protein dynamics in transmembrane receptor signalling

DEFF Research Database (Denmark)

Wang, Yong; Bugge, Katrine Østergaard; Kragelund, Birthe Brandt

2018-01-01

Cells are dependent on transmembrane receptors to communicate and transform chemical and physical signals into intracellular responses. Because receptors transport 'information', conformational changes and protein dynamics play a key mechanistic role. We here review examples where experiment...... to function. Because the receptors function in a heterogeneous environment and need to be able to switch between distinct functional states, they may be particularly sensitive to small perturbations that complicate studies linking dynamics to function....

Use of green fluorescent fusion protein to track activation of the transcription factor osterix during early osteoblast differentiation

International Nuclear Information System (INIS)

Tai Guangping; Christodoulou, Ioannis; Bishop, Anne E.; Polak, Julia M.

2005-01-01

Osterix (Osx) is a transcription factor required for the differentiation of preosteoblasts into fully functioning osteoblasts. However, the pattern of Osx activation during preosteoblast differentiation and maturation has not been clearly defined. Our aim was to study Osx activation during these processes in osteoblasts differentiating from murine and human embryonic stem cells (ESC). To do this, we constructed an Osx-GFP fusion protein reporter system to track Osx translocation within the cells. The distribution of Osx-GFP at representative stages of differentiation was also investigated by screening primary osteoblasts, mesenchymal stem cells, synoviocytes, and pre-adipocytes. Our experiments revealed that Osx-GFP protein was detectable in the cytoplasm of cultured, differentiated ESC 4 days after plating of enzymatically dispersed embryoid bodies. Osterix-GFP protein became translocated into the nucleus on day 7 following transfer of differentiated ESC to osteogenic medium. After 14 days of differentiation, cells showing nuclear translocation of Osx-GFP formed rudimentary bone nodules that continued to increase in number over the following weeks (through day 21). We also found that Osx translocated into the nuclei of mesenchymal stem cells (C3H10T1/2) and pre-osteoblasts (MC3T3-E1) and showed partial activation in pre-adipocytes (MC3T3-L1). These data suggest that Osx activation occurs at a very early point in the differentiation of the mesenchymal-osteoblastic lineage

Synthetic multicellular oscillatory systems: controlling protein dynamics with genetic circuits

International Nuclear Information System (INIS)

Koseska, Aneta; Volkov, Evgenii; Kurths, Juergen

2011-01-01

Synthetic biology is a relatively new research discipline that combines standard biology approaches with the constructive nature of engineering. Thus, recent efforts in the field of synthetic biology have given a perspective to consider cells as 'programmable matter'. Here, we address the possibility of using synthetic circuits to control protein dynamics. In particular, we show how intercellular communication and stochasticity can be used to manipulate the dynamical behavior of a population of coupled synthetic units and, in this manner, finely tune the expression of specific proteins of interest, e.g. in large bioreactors.

Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations.

Science.gov (United States)

Leyva-Leyva, Margarita; López-Díaz, Annia; Barrera, Lourdes; Camacho-Morales, Alberto; Hernandez-Aguilar, Felipe; Carrillo-Casas, Erika M; Arriaga-Pizano, Lourdes; Calderón-Pérez, Jaime; García-Álvarez, Jorge; Orozco-Hoyuela, Gabriel; Piña-Barba, Cristina; Rojas-Martínez, Augusto; Romero-Díaz, Víktor; Lara-Arias, Jorge; Rivera-Bolaños, Nancy; López-Camarillo, César; Moncada-Saucedo, Nidia; Galván-De los Santos, Alejandra; Meza-Urzúa, Fátima; Villarreal-Gómez, Luis; Fuentes-Mera, Lizeth

2015-11-01

Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105(+) and CD105(-)) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105(-) cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated β1-integrin increased in CD105(-) cells during the process. Additionally, the CD105(-) subpopulation showed 3-fold increase of phosphorylated FAK(Y397) compared to CD105(+) cells. Results also indicate that ERK1/2 was activated during CD105(-) bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105(+) differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105(-) showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105(+) showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,(397) and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105(-) subpopulation showed superior adhesion efficiency compared to CD105(+) cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105(-) subpopulation at the maturation stage represents an attractive strategy for clinical use in

Molecular dynamics simulations of protein-tyrosine phosphatase 1B: II. Substrate-enzyme interactions and dynamics

DEFF Research Database (Denmark)

Peters, Günther H.j.; Frimurer, T. M.; Andersen, J. N.

2000-01-01

Molecular dynamics simulations of protein tyrosine phosphatase 1B (PTP1B) complexed with the phosphorylated peptide substrate DADEpYL and the free substrate have been conducted to investigate 1) the physical forces involved in substrate-protein interactions, 2) the importance of enzyme...... to substrate binding. Based on essential dynamics analysis of the PTP1B/DADEpYL trajectory, it is shown that internal motions in the binding pocket occur in a subspace of only a few degrees of freedom. in particular, relatively large flexibilities are observed along several eigenvectors in the segments: Arg(24...... for catalysis. Analysis of the individual enzyme-substrate interaction energies revealed that mainly electrostatic forces contribute to binding. Indeed, calculation of the electrostatic field of the enzyme reveals that only the field surrounding the binding pocket is positive, while the remaining protein...

A SELDI mass spectrometry study of experimental autoimmune encephalomyelitis: sample preparation, reproducibility, and differential protein expression patterns.

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Azzam, Sausan; Broadwater, Laurie; Li, Shuo; Freeman, Ernest J; McDonough, Jennifer; Gregory, Roger B

2013-05-01

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune, inflammatory disease of the central nervous system that is widely used as a model of multiple sclerosis (MS). Mitochondrial dysfunction appears to play a role in the development of neuropathology in MS and may also play a role in disease pathology in EAE. Here, surface enhanced laser desorption ionization mass spectrometry (SELDI-MS) has been employed to obtain protein expression profiles from mitochondrially enriched fractions derived from EAE and control mouse brain. To gain insight into experimental variation, the reproducibility of sub-cellular fractionation, anion exchange fractionation as well as spot-to-spot and chip-to-chip variation using pooled samples from brain tissue was examined. Variability of SELDI mass spectral peak intensities indicates a coefficient of variation (CV) of 15.6% and 17.6% between spots on a given chip and between different chips, respectively. Thinly slicing tissue prior to homogenization with a rotor homogenizer showed better reproducibility (CV = 17.0%) than homogenization of blocks of brain tissue with a Teflon® pestle (CV = 27.0%). Fractionation of proteins with anion exchange beads prior to SELDI-MS analysis gave overall CV values from 16.1% to 18.6%. SELDI mass spectra of mitochondrial fractions obtained from brain tissue from EAE mice and controls displayed 39 differentially expressed proteins (p≤ 0.05) out of a total of 241 protein peaks observed in anion exchange fractions. Hierarchical clustering analysis showed that protein fractions from EAE animals with severe disability clearly segregated from controls. Several components of electron transport chain complexes (cytochrome c oxidase subunit 6b1, subunit 6C, and subunit 4; NADH dehydrogenase flavoprotein 3, alpha subcomplex subunit 2, Fe-S protein 4, and Fe-S protein 6; and ATP synthase subunit e) were identified as possible differentially expressed proteins. Myelin Basic Protein isoform 8 (MBP8) (14.2 k

Translation elicits a growth rate-dependent, genome-wide, differential protein production in Bacillus subtilis.

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Borkowski, Olivier; Goelzer, Anne; Schaffer, Marc; Calabre, Magali; Mäder, Ulrike; Aymerich, Stéphane; Jules, Matthieu; Fromion, Vincent

2016-05-17

Complex regulatory programs control cell adaptation to environmental changes by setting condition-specific proteomes. In balanced growth, bacterial protein abundances depend on the dilution rate, transcript abundances and transcript-specific translation efficiencies. We revisited the current theory claiming the invariance of bacterial translation efficiency. By integrating genome-wide transcriptome datasets and datasets from a library of synthetic gfp-reporter fusions, we demonstrated that translation efficiencies in Bacillus subtilis decreased up to fourfold from slow to fast growth. The translation initiation regions elicited a growth rate-dependent, differential production of proteins without regulators, hence revealing a unique, hard-coded, growth rate-dependent mode of regulation. We combined model-based data analyses of transcript and protein abundances genome-wide and revealed that this global regulation is extensively used in B. subtilis We eventually developed a knowledge-based, three-step translation initiation model, experimentally challenged the model predictions and proposed that a growth rate-dependent drop in free ribosome abundance accounted for the differential protein production. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Mechanical stimulation increases proliferation, differentiation and protein expression in culture

DEFF Research Database (Denmark)

Grossi, Alberto; Yadav, Kavita; Lawson, Moira Ann

2007-01-01

Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. Myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Stimulation due to stretch- or load...... to elucidate also the signaling pathway by which this mechanical stimulation can causes an increase in protein expression. When mechanically stimulated via laminin receptors on cell surface, C(2)C(12) cells showed an increase in cell proliferation and differentiation. Populations undergoing mechanical...... stimulation through laminin receptors show an increase in expression of Myo-D, myogenin and an increase in ERK1/2 phosphorylation. Cells stimulated via fibronectin receptors show no significant increases in fusion competence. We conclude that load induced signalling through integrin containing laminin...

Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein–protein interfaces

International Nuclear Information System (INIS)

Wylie, Benjamin J.; Dzikovski, Boris G.; Pawsey, Shane; Caporini, Marc; Rosay, Melanie; Freed, Jack H.; McDermott, Ann E.

2015-01-01

We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces

Identification of hierarchy of dynamic domains in proteins: comparison of HDWA and HCCP techniques

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Yesylevskyy S. O.

2010-07-01

Full Text Available Aim. There are several techniques for the identification of hierarchy of dynamic domains in proteins. The goal of this work is to compare systematically two recently developed techniques, HCCP and HDWA,on a set of proteins from diverse structural classes. Methods. HDWA and HCCP techniques are used. The HDWA technique is designed to identify hierarchically organized dynamic domains in proteins using the Molecular Dynamics (MD trajectories, while HCCP utilizes the normal modes of simplified elastic network models. Results. It is shown that the dynamic domains found by HDWA are consistent with the domains identified by HCCP and other techniques. At the same time HDWA identifies flexible mobile loops of proteins correctly, which is hard to achieve with other model-based domain identification techniques. Conclusion. HDWA is shown to be a powerful method of analysis of MD trajectories, which can be used in various areas of protein science.

ClC-3 Promotes Osteogenic Differentiation in MC3T3-E1 Cell After Dynamic Compression.

Science.gov (United States)

Wang, Dawei; Wang, Hao; Gao, Feng; Wang, Kun; Dong, Fusheng

2017-06-01

ClC-3 chloride channel has been proved to have a relationship with the expression of osteogenic markers during osteogenesis, persistent static compression can upregulate the expression of ClC-3 and regulate osteodifferentiation in osteoblasts. However, there was no study about the relationship between the expression of ClC-3 and osteodifferentiation after dynamic compression. In this study, we applied dynamic compression on MC3T3-E1 cells to detect the expression of ClC-3, runt-related transcription factor 2 (Runx2), bone morphogenic protein-2 (BMP-2), osteopontin (OPN), nuclear-associated antigen Ki67 (Ki67), and proliferating cell nuclear antigen (PCNA) in biopress system, then we investigated the expression of these genes after dynamic compression with Chlorotoxin (specific ClC-3 chloride channel inhibitor) added. Under transmission electron microscopy, there were more cell surface protrusions, rough surfaced endoplasmic reticulum, mitochondria, Golgi apparatus, abundant glycogen, and lysosomes scattered in the cytoplasm in MC3T3-E1 cells after dynamic compression. The nucleolus was more obvious. We found that ClC-3 was significantly up-regulated after dynamic compression. The compressive force also up-regulated Runx2, BMP-2, and OPN after dynamic compression for 2, 4 and 8 h. The proliferation gene Ki67 and PCNA did not show significantly change after dynamic compression for 8 h. Chlorotoxin did not change the expression of ClC-3 but reduced the expression of Runx2, BMP-2, and OPN after dynamic compression compared with the group without Cltx added. The data from the current study suggested that ClC-3 may promotes osteogenic differentiation in MC3T3-E1 cell after dynamic compression. J. Cell. Biochem. 118: 1606-1613, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Measuring protein dynamics with ultrafast two-dimensional infrared spectroscopy

International Nuclear Information System (INIS)

Adamczyk, Katrin; Candelaresi, Marco; Hunt, Neil T; Robb, Kirsty; Hoskisson, Paul A; Tucker, Nicholas P; Gumiero, Andrea; Walsh, Martin A; Parker, Anthony W

2012-01-01

Recent advances in the methodology and application of ultrafast two-dimensional infrared (2D-IR) spectroscopy to biomolecular systems are reviewed. A description of the 2D-IR technique and the molecular contributions to the observed spectra are presented followed by a discussion of recent literature relating to the use of 2D-IR and associated approaches for measuring protein dynamics. In particular, these include the use of diatomic ligand groups for measuring haem protein dynamics, isotopic labelling strategies and the use of vibrational probe groups. The final section reports on the current state of the art regarding the use of 2D-IR methods to provide insights into biological reaction mechanisms. (topical review)

iTRAQ Protein Profile Differential Analysis of Dormant and Germinated Grassbur Twin Seeds Reveals that Ribosomal Synthesis and Carbohydrate Metabolism Promote Germination Possibly Through the PI3K Pathway.

Science.gov (United States)

Zhang, Guo-Liang; Zhu, Yue; Fu, Wei-Dong; Wang, Peng; Zhang, Rui-Hai; Zhang, Yan-Lei; Song, Zhen; Xia, Gui-Xian; Wu, Jia-He

2016-06-01

Grassbur is a destructive and invasive weed in pastures, and its burs can cause gastric damage to animals. The strong adaptability and reproductive potential of grassbur are partly due to a unique germination mechanism whereby twin seeds develop in a single bur: one seed germinates, but the other remains dormant. To investigate the molecular mechanism of seed germination in twin seeds, we used isobaric tags for relative and absolute quantitation (iTRAQ) to perform a dynamic proteomic analysis of germination and dormancy. A total of 1,984 proteins were identified, 161 of which were considered to be differentially accumulated. The differentially accumulated proteins comprised 102 up-regulated and 59 down-regulated proteins. These proteins were grouped into seven functional categories, ribosomal proteins being the predominant group. The authenticity and accuracy of the results were confirmed by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time reverse transcription-PCR (qPCR). A dynamic proteomic analysis revealed that ribosome synthesis and carbohydrate metabolism affect seed germination possibly through the phosphoinositide 3-kinase (PI3K) pathway. As the PI3K pathway is generally activated by insulin, analyses of seeds treated with exogenous insulin by qPCR, ELISA and iTRAQ confirmed that the PI3K pathway can be activated, which suppresses dormancy and promotes germination in twin grassbur seeds. Together, these results show that the PI3K pathway may play roles in stimulating seed germination in grassbur by modulating ribosomal synthesis and carbohydrate metabolism. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The Role of the Nuclear Envelope Protein MAN1 in Mesenchymal Stem Cell Differentiation.

Science.gov (United States)

Bermeo, Sandra; Al-Saedi, Ahmed; Kassem, Moustapha; Vidal, Christopher; Duque, Gustavo

2017-12-01

Mutations in MAN1, a protein of the nuclear envelope, cause bone phenotypes characterized by hyperostosis. The mechanism of this pro-osteogenic phenotype remains unknown. We increased and decreased MAN1 expression in mesenchymal stem cells (MSC) upon which standard osteogenic and adipogenic differentiation were performed. MAN1 knockdown increased osteogenesis and mineralization. In contrast, osteogenesis remained stable upon MAN1 overexpression. Regarding a mechanism, we found that low levels of MAN1 facilitated the nuclear accumulation of regulatory smads and smads-related complexes, with a concurrently high expression of nuclear β-Catenin. In addition, we found adipogenesis to be decreased in both conditions, although predominantly affected by MAN1 overexpression. Finally, lamin A, a protein of the nuclear envelope that regulates MSC differentiation, was unaffected by changes in MAN1. In conclusion, our studies demonstrated that lower levels of MAN1 in differentiating MSC are associated with higher osteogenesis and lower adipogenesis. High levels of MAN1 only affected adipogenesis. These effects could have an important role in the understanding of the role of the proteins of the nuclear envelope in bone formation. J. Cell. Biochem. 118: 4425-4435, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Application of Solution NMR Spectroscopy to Study Protein Dynamics

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Christoph Göbl

2012-03-01

Full Text Available Recent advances in spectroscopic methods allow the identification of minute fluctuations in a protein structure. These dynamic properties have been identified as keys to some biological processes. The consequences of this structural flexibility can be far‑reaching and they add a new dimension to the structure-function relationship of biomolecules. Nuclear Magnetic Resonance (NMR spectroscopy allows the study of structure as well as dynamics of biomolecules in a very broad range of timescales at atomic level. A number of new NMR methods have been developed recently to allow the measurements of time scales and spatial fluctuations, which in turn provide the thermodynamics associated with the biological processes. Since NMR parameters reflect ensemble measurements, structural ensemble approaches in analyzing NMR data have also been developed. These new methods in some instances can even highlight previously hidden conformational features of the biomolecules. In this review we describe several solution NMR methods to study protein dynamics and discuss their impact on important biological processes.

Comparative Investigation of Normal Modes and Molecular Dynamics of Hepatitis C NS5B Protein

International Nuclear Information System (INIS)

Asafi, M S; Tekpinar, M; Yildirim, A

2016-01-01

Understanding dynamics of proteins has many practical implications in terms of finding a cure for many protein related diseases. Normal mode analysis and molecular dynamics methods are widely used physics-based computational methods for investigating dynamics of proteins. In this work, we studied dynamics of Hepatitis C NS5B protein with molecular dynamics and normal mode analysis. Principal components obtained from a 100 nanoseconds molecular dynamics simulation show good overlaps with normal modes calculated with a coarse-grained elastic network model. Coarse-grained normal mode analysis takes at least an order of magnitude shorter time. Encouraged by this good overlaps and short computation times, we analyzed further low frequency normal modes of Hepatitis C NS5B. Motion directions and average spatial fluctuations have been analyzed in detail. Finally, biological implications of these motions in drug design efforts against Hepatitis C infections have been elaborated. (paper)

Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations.

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Samuel Hertig

2016-06-01

Full Text Available Molecular dynamics (MD simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein's constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery-the fact that the two sites involved influence one another in a symmetrical manner-can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest.

Inhibition of the NAD-dependent protein deacetylase SIRT2 induces granulocytic differentiation in human leukemia cells.

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Yoshitaka Sunami

Full Text Available Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia-retinoic acid receptor α (PML-RAR-α stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation.

Variable-angle epifluorescence microscopy characterizes protein dynamics in the vicinity of plasma membrane in plant cells.

Science.gov (United States)

Chen, Tong; Ji, Dongchao; Tian, Shiping

2018-03-14

The assembly of protein complexes and compositional lipid patterning act together to endow cells with the plasticity required to maintain compositional heterogeneity with respect to individual proteins. Hence, the applications for imaging protein localization and dynamics require high accuracy, particularly at high spatio-temporal level. We provided experimental data for the applications of Variable-Angle Epifluorescence Microscopy (VAEM) in dissecting protein dynamics in plant cells. The VAEM-based co-localization analysis took penetration depth and incident angle into consideration. Besides direct overlap of dual-color fluorescence signals, the co-localization analysis was carried out quantitatively in combination with the methodology for calculating puncta distance and protein proximity index. Besides, simultaneous VAEM tracking of cytoskeletal dynamics provided more insights into coordinated responses of actin filaments and microtubules. Moreover, lateral motility of membrane proteins was analyzed by calculating diffusion coefficients and kymograph analysis, which represented an alternative method for examining protein motility. The present study presented experimental evidence on illustrating the use of VAEM in tracking and dissecting protein dynamics, dissecting endosomal dynamics, cell structure assembly along with membrane microdomain and protein motility in intact plant cells.

Dynamic prestress in a globular protein.

Directory of Open Access Journals (Sweden)

Scott A Edwards

Full Text Available A protein at equilibrium is commonly thought of as a fully relaxed structure, with the intra-molecular interactions showing fluctuations around their energy minimum. In contrast, here we find direct evidence for a protein as a molecular tensegrity structure, comprising a balance of tensed and compressed interactions, a concept that has been put forward for macroscopic structures. We quantified the distribution of inter-residue prestress in ubiquitin and immunoglobulin from all-atom molecular dynamics simulations. The network of highly fluctuating yet significant inter-residue forces in proteins is a consequence of the intrinsic frustration of a protein when sampling its rugged energy landscape. In beta sheets, this balance of forces is found to compress the intra-strand hydrogen bonds. We estimate that the observed magnitude of this pre-compression is enough to induce significant changes in the hydrogen bond lifetimes; thus, prestress, which can be as high as a few 100 pN, can be considered a key factor in determining the unfolding kinetics and pathway of proteins under force. Strong pre-tension in certain salt bridges on the other hand is connected to the thermodynamic stability of ubiquitin. Effective force profiles between some side-chains reveal the signature of multiple, distinct conformational states, and such static disorder could be one factor explaining the growing body of experiments revealing non-exponential unfolding kinetics of proteins. The design of prestress distributions in engineering proteins promises to be a new tool for tailoring the mechanical properties of made-to-order nanomaterials.

Application of partial differential equation modeling of the control/structural dynamics of flexible spacecraft

Science.gov (United States)

Taylor, Lawrence W., Jr.; Rajiyah, H.

1991-01-01

Partial differential equations for modeling the structural dynamics and control systems of flexible spacecraft are applied here in order to facilitate systems analysis and optimization of these spacecraft. Example applications are given, including the structural dynamics of SCOLE, the Solar Array Flight Experiment, the Mini-MAST truss, and the LACE satellite. The development of related software is briefly addressed.

A Comprehensive Analysis of Chromoplast Differentiation Reveals Complex Protein Changes Associated with Plastoglobule Biogenesis and Remodeling of Protein Systems in Sweet Orange Flesh1[OPEN

Science.gov (United States)

Wang, Lun; Deng, Xiuxin

2015-01-01

Globular and crystalloid chromoplasts were observed to be region specifically formed in sweet orange (Citrus sinensis) flesh and converted from amyloplasts during fruit maturation, which was associated with the composition of specific carotenoids and the expression of carotenogenic genes. Subsequent isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analyses of purified plastids from the flesh during chromoplast differentiation and senescence identified 1,386 putative plastid-localized proteins, 1,016 of which were quantified by spectral counting. The iTRAQ values reflecting the expression abundance of three identified proteins were validated by immunoblotting. Based on iTRAQ data, chromoplastogenesis appeared to be associated with three major protein expression patterns: (1) marked decrease in abundance of the proteins participating in the translation machinery through ribosome assembly; (2) increase in abundance of the proteins involved in terpenoid biosynthesis (including carotenoids), stress responses (redox, ascorbate, and glutathione), and development; and (3) maintenance of the proteins for signaling and DNA and RNA. Interestingly, a strong increase in abundance of several plastoglobule-localized proteins coincided with the formation of plastoglobules in the chromoplast. The proteomic data also showed that stable functioning of protein import, suppression of ribosome assembly, and accumulation of chromoplast proteases are correlated with the amyloplast-to-chromoplast transition; thus, these processes may play a collective role in chromoplast biogenesis and differentiation. By contrast, the chromoplast senescence process was inferred to be associated with significant increases in stress response and energy supply. In conclusion, this comprehensive proteomic study identified many potentially new plastid-localized proteins and provides insights into the potential developmental and molecular mechanisms underlying chromoplast

A stochastic differential equation framework for the timewise dynamics of turbulent velocities

DEFF Research Database (Denmark)

Barndorff-Nielsen, Ole Eiler; Schmiegel, Jürgen

2008-01-01

We discuss a stochastic differential equation as a modeling framework for the timewise dynamics of turbulent velocities. The equation is capable of capturing basic stylized facts of the statistics of temporal velocity increments. In particular, we focus on the evolution of the probability density...

Activation of protein kinase A and exchange protein directly activated by cAMP promotes adipocyte differentiation of human mesenchymal stem cells

DEFF Research Database (Denmark)

Jia, Bingbing; Madsen, Lise; Petersen, Rasmus Koefoed

2012-01-01

) and exchange protein directly activated by cAMP (Epac) in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS). We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence......Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA...... results emphasize the need for cAMP signaling in concert with treatment with a PPARγ or PPARδ agonist to secure efficient adipocyte differentiation of human hMADS mesenchymal stem cells....

Comparative proteomic analysis of differentially expressed proteins in the urine of reservoir hosts of leptospirosis.

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Jarlath E Nally

Full Text Available Rattus norvegicus is a natural reservoir host for pathogenic species of Leptospira. Experimentally infected rats remain clinically normal, yet persistently excrete large numbers of leptospires from colonized renal tubules via urine, despite a specific host immune response. Whilst persistent renal colonization and shedding is facilitated in part by differential antigen expression by leptospires to evade host immune responses, there is limited understanding of kidney and urinary proteins expressed by the host that facilitates such biological equilibrium. Urine pellets were collected from experimentally infected rats shedding leptospires and compared to urine from non-infected controls spiked with in vitro cultivated leptospires for analysis by 2-D DIGE. Differentially expressed host proteins include membrane metallo endopeptidase, napsin A aspartic peptidase, vacuolar H+ATPase, kidney aminopeptidase and immunoglobulin G and A. Loa22, a virulence factor of Leptospira, as well as the GroEL, were increased in leptospires excreted in urine compared to in vitro cultivated leptospires. Urinary IgG from infected rats was specific for leptospires. Results confirm differential protein expression by both host and pathogen during chronic disease and include markers of kidney function and immunoglobulin which are potential biomarkers of infection.

Distance-Based Configurational Entropy of Proteins from Molecular Dynamics Simulations.

Science.gov (United States)

Fogolari, Federico; Corazza, Alessandra; Fortuna, Sara; Soler, Miguel Angel; VanSchouwen, Bryan; Brancolini, Giorgia; Corni, Stefano; Melacini, Giuseppe; Esposito, Gennaro

2015-01-01

Estimation of configurational entropy from molecular dynamics trajectories is a difficult task which is often performed using quasi-harmonic or histogram analysis. An entirely different approach, proposed recently, estimates local density distribution around each conformational sample by measuring the distance from its nearest neighbors. In this work we show this theoretically well grounded the method can be easily applied to estimate the entropy from conformational sampling. We consider a set of systems that are representative of important biomolecular processes. In particular: reference entropies for amino acids in unfolded proteins are obtained from a database of residues not participating in secondary structure elements;the conformational entropy of folding of β2-microglobulin is computed from molecular dynamics simulations using reference entropies for the unfolded state;backbone conformational entropy is computed from molecular dynamics simulations of four different states of the EPAC protein and compared with order parameters (often used as a measure of entropy);the conformational and rototranslational entropy of binding is computed from simulations of 20 tripeptides bound to the peptide binding protein OppA and of β2-microglobulin bound to a citrate coated gold surface. This work shows the potential of the method in the most representative biological processes involving proteins, and provides a valuable alternative, principally in the shown cases, where other approaches are problematic.

Protein Charge and Mass Contribute to the Spatio-temporal Dynamics of Protein-Protein Interactions in a Minimal Proteome

Science.gov (United States)

Xu, Yu; Wang, Hong; Nussinov, Ruth; Ma, Buyong

2013-01-01

We constructed and simulated a ‘minimal proteome’ model using Langevin dynamics. It contains 206 essential protein types which were compiled from the literature. For comparison, we generated six proteomes with randomized concentrations. We found that the net charges and molecular weights of the proteins in the minimal genome are not random. The net charge of a protein decreases linearly with molecular weight, with small proteins being mostly positively charged and large proteins negatively charged. The protein copy numbers in the minimal genome have the tendency to maximize the number of protein-protein interactions in the network. Negatively charged proteins which tend to have larger sizes can provide large collision cross-section allowing them to interact with other proteins; on the other hand, the smaller positively charged proteins could have higher diffusion speed and are more likely to collide with other proteins. Proteomes with random charge/mass populations form less stable clusters than those with experimental protein copy numbers. Our study suggests that ‘proper’ populations of negatively and positively charged proteins are important for maintaining a protein-protein interaction network in a proteome. It is interesting to note that the minimal genome model based on the charge and mass of E. Coli may have a larger protein-protein interaction network than that based on the lower organism M. pneumoniae. PMID:23420643

Non-genetic heterogeneity, criticality and cell differentiation.

Science.gov (United States)

Pal, Mainak; Ghosh, Sayantari; Bose, Indrani

2014-11-27

The different cell types in a living organism acquire their identity through the process of cell differentiation in which multipotent progenitor cells differentiate into distinct cell types. Experimental evidence and analysis of large-scale microarray data establish the key role played by a two-gene motif in cell differentiation in a number of cell systems. The two genes express transcription factors which repress each other's expression and autoactivate their own production. A number of theoretical models have recently been proposed based on the two-gene motif to provide a physical understanding of how cell differentiation occurs. In this paper, we study a simple model of cell differentiation which assumes no cooperativity in the regulation of gene expression by the transcription factors. The latter repress each other's activity directly through DNA binding and indirectly through the formation of heterodimers. We specifically investigate how deterministic processes combined with stochasticity contribute in bringing about cell differentiation. The deterministic dynamics of our model give rise to a supercritical pitchfork bifurcation from an undifferentiated stable steady state to two differentiated stable steady states. The stochastic dynamics of our model are studied using the approaches based on the Langevin equations and the linear noise approximation. The simulation results provide a new physical understanding of recent experimental observations. We further propose experimental measurements of quantities like the variance and the lag-1 autocorrelation function in protein fluctuations as the early signatures of an approaching bifurcation point in the cell differentiation process.

Lipid Regulated Intramolecular Conformational Dynamics of SNARE-Protein Ykt6

Science.gov (United States)

Dai, Yawei; Seeger, Markus; Weng, Jingwei; Song, Song; Wang, Wenning; Tan, Yan-Wen

2016-08-01

Cellular informational and metabolic processes are propagated with specific membrane fusions governed by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNARE). SNARE protein Ykt6 is highly expressed in brain neurons and plays a critical role in the membrane-trafficking process. Studies suggested that Ykt6 undergoes a conformational change at the interface between its longin domain and the SNARE core. In this work, we study the conformational state distributions and dynamics of rat Ykt6 by means of single-molecule Förster Resonance Energy Transfer (smFRET) and Fluorescence Cross-Correlation Spectroscopy (FCCS). We observed that intramolecular conformational dynamics between longin domain and SNARE core occurred at the timescale ~200 μs. Furthermore, this dynamics can be regulated and even eliminated by the presence of lipid dodecylphoshpocholine (DPC). Our molecular dynamic (MD) simulations have shown that, the SNARE core exhibits a flexible structure while the longin domain retains relatively stable in apo state. Combining single molecule experiments and theoretical MD simulations, we are the first to provide a quantitative dynamics of Ykt6 and explain the functional conformational change from a qualitative point of view.

Sparse Additive Ordinary Differential Equations for Dynamic Gene Regulatory Network Modeling.

Science.gov (United States)

Wu, Hulin; Lu, Tao; Xue, Hongqi; Liang, Hua

2014-04-02

The gene regulation network (GRN) is a high-dimensional complex system, which can be represented by various mathematical or statistical models. The ordinary differential equation (ODE) model is one of the popular dynamic GRN models. High-dimensional linear ODE models have been proposed to identify GRNs, but with a limitation of the linear regulation effect assumption. In this article, we propose a sparse additive ODE (SA-ODE) model, coupled with ODE estimation methods and adaptive group LASSO techniques, to model dynamic GRNs that could flexibly deal with nonlinear regulation effects. The asymptotic properties of the proposed method are established and simulation studies are performed to validate the proposed approach. An application example for identifying the nonlinear dynamic GRN of T-cell activation is used to illustrate the usefulness of the proposed method.

Shedding Light on Protein Folding, Structural and Functional Dynamics by Single Molecule Studies

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Krutika Bavishi

2014-11-01

Full Text Available The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements. Single molecule studies have thus provided novel insights about how the dynamic sampling of the free energy landscape dictates all aspects of protein behavior; from its folding to function. Here we will survey some of the state of the art contributions in deciphering mechanisms that underlie protein folding, structural and functional dynamics by single molecule fluorescence microscopy techniques. We will discuss a few selected examples highlighting the power of the emerging techniques and finally discuss the future improvements and directions.

Identification of differentially expressed proteins in spontaneous thymic lymphomas from knockout mice with deletion of p53

DEFF Research Database (Denmark)

Honoré, Bent; Buus, Søren; Claësson, Mogens H

2008-01-01

ABSTRACT: BACKGROUND: Knockout mice with a deletion of p53 spontaneously develop thymic lymphomas. Two cell lines (SM5 and SM7), established from two independent tumours, exhibited about fifty to seventy two-fold differentially expressed proteins compared to wild type thymocytes by two-dimensiona......ABSTRACT: BACKGROUND: Knockout mice with a deletion of p53 spontaneously develop thymic lymphomas. Two cell lines (SM5 and SM7), established from two independent tumours, exhibited about fifty to seventy two-fold differentially expressed proteins compared to wild type thymocytes by two...... alpha type 3, transforming acidic coiled-coil containing protein 3, mitochondrial ornithine aminotransferase and epidermal fatty acid binding protein and down-regulation of adenylosuccinate synthetase, tubulin beta-3 chain, a 25 kDa actin fragment, proteasome subunit beta type 9, cofilin-1 and glia...

Periodic solutions of first-order functional differential equations in population dynamics

CERN Document Server

Padhi, Seshadev; Srinivasu, P D N

2014-01-01

This book provides cutting-edge results on the existence of multiple positive periodic solutions of first-order functional differential equations. It demonstrates how the Leggett-Williams fixed-point theorem can be applied to study the existence of two or three positive periodic solutions of functional differential equations with real-world applications, particularly with regard to the Lasota-Wazewska model, the Hematopoiesis model, the Nicholsons Blowflies model, and some models with Allee effects. Many interesting sufficient conditions are given for the dynamics that include nonlinear characteristics exhibited by population models. The last chapter provides results related to the global appeal of solutions to the models considered in the earlier chapters. The techniques used in this book can be easily understood by anyone with a basic knowledge of analysis. This book offers a valuable reference guide for students and researchers in the field of differential equations with applications to biology, ecology, a...

Rare sugar D-allose strongly induces thioredoxin-interacting protein and inhibits osteoclast differentiation in Raw264 cells.

Science.gov (United States)

Yamada, Kana; Noguchi, Chisato; Kamitori, Kazuyo; Dong, Youyi; Hirata, Yuko; Hossain, Mohammad A; Tsukamoto, Ikuko; Tokuda, Masaaki; Yamaguchi, Fuminori

2012-02-01

Oxidative stress modulates the osteoclast differentiation via redox systems, and thioredoxin 1 (Trx) promotes the osteoclast formation by regulating the activity of transcription factors. The function of Trx is known to be regulated by its binding partner, thioredoxin-interacting protein (TXNIP). We previously reported that the expression of TXNIP gene is strongly induced by a rare sugar D-allose. In this study, we tested the hypothesis that D-allose could inhibit the osteoclast differentiation by regulating the Trx function. We used a murine Raw264 cell line that differentiates to the osteoclast by the receptor activator of nuclear factor-κB ligand (RANKL) treatment. The effect of sugars was evaluated by tartrate-resistant acid phosphatase staining. The expression and localization of TXNIP and Trx protein were examined by Western blotting and immunohistochemisty. The activity of the nuclear factor-κB, nuclear factor of activated T cells, and activator protein 1 transcription factors was measured by the luciferase reporter assay. The addition of D-allose (25 mmol/L) inhibited the osteoclast differentiation down to 9.53% ± 1.27% of a receptor activator of nuclear factor-κB ligand-only treatment. During the osteoclast differentiation, a significant increase of TNXIP was observed by D-allose treatment. The immunohistochemical analysis showed that both Trx and TXNIP existed in the nucleus in preosteoclasts and osteoclasts. Overexpression of TXNIP by plasmid transfection also inhibited the osteoclast formation, indicating the functional importance of TXNIP for the osteoclast differentiation. Transcriptional activity of the activator protein 1, nuclear factor-κB, and nuclear factor of activated T cells, known to be modulated by Trx, were inhibited by D-allose. In conclusion, our data indicate that D-allose is a strong inhibitor of the osteoclast differentiation, and this effect could be caused by TXNIP induction and a resulting inhibition of the Trx function

A computational investigation on the connection between dynamics properties of ribosomal proteins and ribosome assembly.

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Brittany Burton

Full Text Available Assembly of the ribosome from its protein and RNA constituents has been studied extensively over the past 50 years, and experimental evidence suggests that prokaryotic ribosomal proteins undergo conformational changes during assembly. However, to date, no studies have attempted to elucidate these conformational changes. The present work utilizes computational methods to analyze protein dynamics and to investigate the linkage between dynamics and binding of these proteins during the assembly of the ribosome. Ribosomal proteins are known to be positively charged and we find the percentage of positive residues in r-proteins to be about twice that of the average protein: Lys+Arg is 18.7% for E. coli and 21.2% for T. thermophilus. Also, positive residues constitute a large proportion of RNA contacting residues: 39% for E. coli and 46% for T. thermophilus. This affirms the known importance of charge-charge interactions in the assembly of the ribosome. We studied the dynamics of three primary proteins from E. coli and T. thermophilus 30S subunits that bind early in the assembly (S15, S17, and S20 with atomic molecular dynamic simulations, followed by a study of all r-proteins using elastic network models. Molecular dynamics simulations show that solvent-exposed proteins (S15 and S17 tend to adopt more stable solution conformations than an RNA-embedded protein (S20. We also find protein residues that contact the 16S rRNA are generally more mobile in comparison with the other residues. This is because there is a larger proportion of contacting residues located in flexible loop regions. By the use of elastic network models, which are computationally more efficient, we show that this trend holds for most of the 30S r-proteins.

Protein Dynamics in the Plant Extracellular Space

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Leonor Guerra-Guimarães

2016-07-01

Full Text Available The extracellular space (ECS or apoplast is the plant cell compartment external to the plasma membrane, which includes the cell walls, the intercellular space and the apoplastic fluid (APF. The present review is focused on APF proteomics papers and intends to draw information on the metabolic processes occurring in the ECS under abiotic and biotic stresses, as well as under non-challenged conditions. The large majority of the proteins detected are involved in “cell wall organization and biogenesis”, “response to stimulus” and “protein metabolism”. It becomes apparent that some proteins are always detected, irrespective of the experimental conditions, although with different relative contribution. This fact suggests that non-challenged plants have intrinsic constitutive metabolic processes of stress/defense in the ECS. In addition to the multiple functions ascribed to the ECS proteins, should be considered the interactions established between themselves and with the plasma membrane and its components. These interactions are crucial in connecting exterior and interior of the cell, and even simple protein actions in the ECS can have profound effects on plant performance. The proteins of the ECS are permanently contributing to the high dynamic nature of this plant compartment, which seems fundamental to plant development and adaptation to the environmental conditions.

Identification of differentially expressed proteins in vitamin B 12

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Swati Varshney

2015-01-01

Full Text Available Background: Vitamin B 12 (cobalamin is a water-soluble vitamin generally synthesized by microorganisms. Mammals cannot synthesize this vitamin but have evolved processes for absorption, transport and cellular uptake of this vitamin. Only about 30% of vitamin B 12 , which is bound to the protein transcobalamin (TC (Holo-TC [HoloTC] enters into the cell and hence is referred to as the biologically active form of vitamin B 12 . Vitamin B 12 deficiency leads to several complex disorders, including neurological disorders and anemia. We had earlier shown that vitamin B 12 deficiency is associated with coronary artery disease (CAD in Indian population. In the current study, using a proteomics approach we identified proteins that are differentially expressed in the plasma of individuals with low HoloTC levels. Materials and Methods: We used isobaric-tagging method of relative and absolute quantitation to identify proteins that are differently expressed in individuals with low HoloTC levels when compared to those with normal HoloTC level. Results: In two replicate isobaric tags for relative and absolute quantitation experiments several proteins involved in lipid metabolism, blood coagulation, cholesterol metabolic process, and lipoprotein metabolic process were found to be altered in individuals having low HoloTC levels. Conclusions: Our study indicates that low HoloTc levels could be a risk factor in the development of CAD.

Protein Availability and Satellite Cell Dynamics in Skeletal Muscle.

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Shamim, Baubak; Hawley, John A; Camera, Donny M

2018-06-01

Human skeletal muscle satellite cells are activated in response to both resistance and endurance exercise. It was initially proposed that satellite cell proliferation and differentiation were only required to support resistance exercise-induced hypertrophy. However, satellite cells may also play a role in muscle fibre remodelling after endurance-based exercise and extracellular matrix regulation. Given the importance of dietary protein, particularly branched chain amino acids, in supporting myofibrillar and mitochondrial adaptations to both resistance and endurance-based training, a greater understanding of how protein intake impacts satellite cell activity would provide further insight into the mechanisms governing skeletal muscle remodelling with exercise. While many studies have investigated the capacity for protein ingestion to increase post-exercise rates of muscle protein synthesis, few investigations have examined the role for protein ingestion to modulate satellite cell activity. Here we review the molecular mechanisms controlling the activation of satellite cells in response to mechanical stress and protein intake in both in vitro and in vivo models. We provide a mechanistic framework that describes how protein ingestion may enhance satellite activity and promote exercise adaptations in human skeletal muscle.

Reverse micelles as a tool for probing solvent modulation of protein dynamics: Reverse micelle encapsulated hemoglobin

Science.gov (United States)

Roche, Camille J.; Dantsker, David; Heller, Elizabeth R.; Sabat, Joseph E.; Friedman, Joel M.

2013-08-01

Hydration waters impact protein dynamics. Dissecting the interplay between hydration waters and dynamics requires a protein that manifests a broad range of dynamics. Proteins in reverse micelles (RMs) have promise as tools to achieve this objective because the water content can be manipulated. Hemoglobin is an appropriate tool with which to probe hydration effects. We describe both a protocol for hemoglobin encapsulation in reverse micelles and a facile method using PEG and cosolvents to manipulate water content. Hydration properties are probed using the water-sensitive fluorescence from Hb bound pyranine and covalently attached Badan. Protein dynamics are probed through ligand recombination traces derived from photodissociated carbonmonoxy hemoglobin on a log scale that exposes the potential role of both α and β solvent fluctuations in modulating protein dynamics. The results open the possibility of probing hydration level phenomena in this system using a combination of NMR and optical probes.

Watching proteins function with picosecond X-ray crystallography and molecular dynamics simulations.

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Anfinrud, Philip

2006-03-01

Time-resolved electron density maps of myoglobin, a ligand-binding heme protein, have been stitched together into movies that unveil with molecular dynamics (MD) calculations and picosecond time-resolved X-ray structures provides single-molecule insights into mechanisms of protein function. Ensemble-averaged MD simulations of the L29F mutant of myoglobin following ligand dissociation reproduce the direction, amplitude, and timescales of crystallographically-determined structural changes. This close agreement with experiments at comparable resolution in space and time validates the individual MD trajectories, which identify and structurally characterize a conformational switch that directs dissociated ligands to one of two nearby protein cavities. This unique combination of simulation and experiment unveils functional protein motions and illustrates at an atomic level relationships among protein structure, dynamics, and function. In collaboration with Friedrich Schotte and Gerhard Hummer, NIH.

Local dynamics of proteins and DNA evaluated from crystallographic B factors

International Nuclear Information System (INIS)

Schneider, Bohdan; Gelly, Jean-Christophe; Brevern, Alexandre G. de; Černý, Jiří

2014-01-01

Distributions of scaled B factors from 704 protein–DNA complexes reflect primarily the neighbourhood of amino-acid and nucleotide residues: their flexibility grows from the protein core to protein–protein and protein–DNA interfaces, to solvent-exposed residues. Some of the findings clearly observed at higher resolution structures can no longer be observed for structures at low resolution indicating problems in refinement protocols. The dynamics of protein and nucleic acid structures is as important as their average static picture. The local molecular dynamics concealed in diffraction images is expressed as so-called B factors. To find out how the crystal-derived B factors represent the dynamic behaviour of atoms and residues of proteins and DNA in their complexes, the distributions of scaled B factors from a carefully curated data set of over 700 protein–DNA crystal structures were analyzed [Schneider et al. (2014 ▶), Nucleic Acids Res.42, 3381–3394]. Amino acids and nucleotides were categorized based on their molecular neighbourhood as solvent-accessible, solvent-inaccessible (i.e. forming the protein core) or lying at protein–protein or protein–DNA interfaces; the backbone and side-chain atoms were analyzed separately. The B factors of two types of crystal-ordered water molecules were also analyzed. The analysis confirmed several expected features of protein and DNA dynamics, but also revealed surprising facts. Solvent-accessible amino acids have B factors that are larger than those of residues at the biomolecular interfaces, and core-forming amino acids are the most restricted in their movement. A unique feature of the latter group is that their side-chain and backbone atoms are restricted in their movement to the same extent; in all other amino-acid groups the side chains are more floppy than the backbone. The low values of the B factors of water molecules bridging proteins with DNA and the very large fluctuations of DNA phosphates are

Local dynamics of proteins and DNA evaluated from crystallographic B factors

Energy Technology Data Exchange (ETDEWEB)

Schneider, Bohdan, E-mail: bohdan.schneider@gmail.com [Institute of Biotechnology AS CR, Videnska 1083, 142 20 Prague (Czech Republic); Gelly, Jean-Christophe; Brevern, Alexandre G. de [INSERM, U1134, DSIMB, 75739 Paris (France); Université Paris Diderot, Sorbonne Paris Cité, UMR-S 1134, 75739 Paris (France); Institut National de la Transfusion Sanguine (INTS), 75739 Paris (France); Laboratoire d’Excellence GR-Ex, 75739 Paris (France); Černý, Jiří [Institute of Biotechnology AS CR, Videnska 1083, 142 20 Prague (Czech Republic)

2014-09-01

Distributions of scaled B factors from 704 protein–DNA complexes reflect primarily the neighbourhood of amino-acid and nucleotide residues: their flexibility grows from the protein core to protein–protein and protein–DNA interfaces, to solvent-exposed residues. Some of the findings clearly observed at higher resolution structures can no longer be observed for structures at low resolution indicating problems in refinement protocols. The dynamics of protein and nucleic acid structures is as important as their average static picture. The local molecular dynamics concealed in diffraction images is expressed as so-called B factors. To find out how the crystal-derived B factors represent the dynamic behaviour of atoms and residues of proteins and DNA in their complexes, the distributions of scaled B factors from a carefully curated data set of over 700 protein–DNA crystal structures were analyzed [Schneider et al. (2014 ▶), Nucleic Acids Res.42, 3381–3394]. Amino acids and nucleotides were categorized based on their molecular neighbourhood as solvent-accessible, solvent-inaccessible (i.e. forming the protein core) or lying at protein–protein or protein–DNA interfaces; the backbone and side-chain atoms were analyzed separately. The B factors of two types of crystal-ordered water molecules were also analyzed. The analysis confirmed several expected features of protein and DNA dynamics, but also revealed surprising facts. Solvent-accessible amino acids have B factors that are larger than those of residues at the biomolecular interfaces, and core-forming amino acids are the most restricted in their movement. A unique feature of the latter group is that their side-chain and backbone atoms are restricted in their movement to the same extent; in all other amino-acid groups the side chains are more floppy than the backbone. The low values of the B factors of water molecules bridging proteins with DNA and the very large fluctuations of DNA phosphates are

ESTIMATION OF CONSTANT AND TIME-VARYING DYNAMIC PARAMETERS OF HIV INFECTION IN A NONLINEAR DIFFERENTIAL EQUATION MODEL.

Science.gov (United States)

Liang, Hua; Miao, Hongyu; Wu, Hulin

2010-03-01

Modeling viral dynamics in HIV/AIDS studies has resulted in deep understanding of pathogenesis of HIV infection from which novel antiviral treatment guidance and strategies have been derived. Viral dynamics models based on nonlinear differential equations have been proposed and well developed over the past few decades. However, it is quite challenging to use experimental or clinical data to estimate the unknown parameters (both constant and time-varying parameters) in complex nonlinear differential equation models. Therefore, investigators usually fix some parameter values, from the literature or by experience, to obtain only parameter estimates of interest from clinical or experimental data. However, when such prior information is not available, it is desirable to determine all the parameter estimates from data. In this paper, we intend to combine the newly developed approaches, a multi-stage smoothing-based (MSSB) method and the spline-enhanced nonlinear least squares (SNLS) approach, to estimate all HIV viral dynamic parameters in a nonlinear differential equation model. In particular, to the best of our knowledge, this is the first attempt to propose a comparatively thorough procedure, accounting for both efficiency and accuracy, to rigorously estimate all key kinetic parameters in a nonlinear differential equation model of HIV dynamics from clinical data. These parameters include the proliferation rate and death rate of uninfected HIV-targeted cells, the average number of virions produced by an infected cell, and the infection rate which is related to the antiviral treatment effect and is time-varying. To validate the estimation methods, we verified the identifiability of the HIV viral dynamic model and performed simulation studies. We applied the proposed techniques to estimate the key HIV viral dynamic parameters for two individual AIDS patients treated with antiretroviral therapies. We demonstrate that HIV viral dynamics can be well characterized and

Evaluation of dynamic enhanced CT scanning in the differentiation of adrenal lipid-poor adenomas with metastases

International Nuclear Information System (INIS)

Fang Xiangming; Hu Chunhong; Hu Xiaoyun; Chen Hongwei; Wu Liyuan; Zou Xinnong; Qian Pingyan

2006-01-01

Objective: To evaluate dynamic enhanced CT in differentiating adrenal metastases from adrenal lipid-poor adenomas(ALPA). Methods: Both plain and dynamic enhanced CT scanning was performed in 9 metastases with 13 masses and 28 lipid-poor adenoma with 30 masses. The types of time-density curve according to peak time(PT) and relative washout percentage(Washr) besides shape, size, margin, internal structure, surrounding status and enhanced pattern of each lesion were measuerd and compared between the two groups of metastases and ALPA. Results: There is difference between metastases and ALPA in the aspects of shape, density, neighboring structure and the type of enhancement. The type of TDC of matastases was characterized by fast-washin and fast-washout, which was quite differed from the type of TDC of ALPA characterized by fast-washin and slow-washout. According to this, the sensitiveity and specificity for differentiating metastases from ALPA were 96.7%, 92.3%. Conclusion: The types of TDC of dynamic enhanced CT is of great value in differentiating metastases from ALPA. (authors)

Conformational dynamics of amyloid proteins at the aqueous interface

Science.gov (United States)

Armbruster, Matthew; Horst, Nathan; Aoki, Brendy; Malik, Saad; Soto, Patricia

2013-03-01

Amyloid proteins is a class of proteins that exhibit distinct monomeric and oligomeric conformational states hallmark of deleterious neurological diseases for which there are not yet cures. Our goal is to examine the extent of which the aqueous/membrane interface modulates the folding energy landscape of amyloid proteins. To this end, we probe the dynamic conformational ensemble of amyloids (monomer prion protein and Alzheimer's Ab protofilaments) interacting with model bilayers. We will present the results of our coarse grain molecular modeling study in terms of the existence of preferential binding spots of the amyloid to the bilayer and the response of the bilayer to the interaction with the amyloid. NSF Nebraska EPSCoR First Award

Dynamical analysis of yeast protein interaction network during the sake brewing process.

Science.gov (United States)

Mirzarezaee, Mitra; Sadeghi, Mehdi; Araabi, Babak N

2011-12-01

Proteins interact with each other for performing essential functions of an organism. They change partners to get involved in various processes at different times or locations. Studying variations of protein interactions within a specific process would help better understand the dynamic features of the protein interactions and their functions. We studied the protein interaction network of Saccharomyces cerevisiae (yeast) during the brewing of Japanese sake. In this process, yeast cells are exposed to several stresses. Analysis of protein interaction networks of yeast during this process helps to understand how protein interactions of yeast change during the sake brewing process. We used gene expression profiles of yeast cells for this purpose. Results of our experiments revealed some characteristics and behaviors of yeast hubs and non-hubs and their dynamical changes during the brewing process. We found that just a small portion of the proteins (12.8 to 21.6%) is responsible for the functional changes of the proteins in the sake brewing process. The changes in the number of edges and hubs of the yeast protein interaction networks increase in the first stages of the process and it then decreases at the final stages.

Constant pH molecular dynamics of proteins in explicit solvent with proton tautomerism.

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Goh, Garrett B; Hulbert, Benjamin S; Zhou, Huiqing; Brooks, Charles L

2014-07-01

pH is a ubiquitous regulator of biological activity, including protein-folding, protein-protein interactions, and enzymatic activity. Existing constant pH molecular dynamics (CPHMD) models that were developed to address questions related to the pH-dependent properties of proteins are largely based on implicit solvent models. However, implicit solvent models are known to underestimate the desolvation energy of buried charged residues, increasing the error associated with predictions that involve internal ionizable residue that are important in processes like hydrogen transport and electron transfer. Furthermore, discrete water and ions cannot be modeled in implicit solvent, which are important in systems like membrane proteins and ion channels. We report on an explicit solvent constant pH molecular dynamics framework based on multi-site λ-dynamics (CPHMD(MSλD)). In the CPHMD(MSλD) framework, we performed seamless alchemical transitions between protonation and tautomeric states using multi-site λ-dynamics, and designed novel biasing potentials to ensure that the physical end-states are predominantly sampled. We show that explicit solvent CPHMD(MSλD) simulations model realistic pH-dependent properties of proteins such as the Hen-Egg White Lysozyme (HEWL), binding domain of 2-oxoglutarate dehydrogenase (BBL) and N-terminal domain of ribosomal protein L9 (NTL9), and the pKa predictions are in excellent agreement with experimental values, with a RMSE ranging from 0.72 to 0.84 pKa units. With the recent development of the explicit solvent CPHMD(MSλD) framework for nucleic acids, accurate modeling of pH-dependent properties of both major class of biomolecules-proteins and nucleic acids is now possible. © 2013 Wiley Periodicals, Inc.

Complexes of γ-tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells

International Nuclear Information System (INIS)

Kukharskyy, Vitaliy; Sulimenko, Vadym; Macurek, Libor; Sulimenko, Tetyana; Draberova, Eduarda; Draber, Pavel

2004-01-01

Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that γ-tubulin (γ-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, γ-tubulin, and with anti-phosphotyrosine antibody revealed that γ-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in γ-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated γ-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing γ-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of γ-tubulin interaction with tubulin dimers or other proteins during neurogenesis

Transcriptional regulation by Polycomb group proteins

DEFF Research Database (Denmark)

Di Croce, Luciano; Helin, Kristian

2013-01-01

Polycomb group (PcG) proteins are epigenetic regulators of transcription that have key roles in stem-cell identity, differentiation and disease. Mechanistically, they function within multiprotein complexes, called Polycomb repressive complexes (PRCs), which modify histones (and other proteins......) and silence target genes. The dynamics of PRC1 and PRC2 components has been the focus of recent research. Here we discuss our current knowledge of the PRC complexes, how they are targeted to chromatin and how the high diversity of the PcG proteins allows these complexes to influence cell identity....

A simple quantitative model of macromolecular crowding effects on protein folding: Application to the murine prion protein(121-231)

Science.gov (United States)

Bergasa-Caceres, Fernando; Rabitz, Herschel A.

2013-06-01

A model of protein folding kinetics is applied to study the effects of macromolecular crowding on protein folding rate and stability. Macromolecular crowding is found to promote a decrease of the entropic cost of folding of proteins that produces an increase of both the stability and the folding rate. The acceleration of the folding rate due to macromolecular crowding is shown to be a topology-dependent effect. The model is applied to the folding dynamics of the murine prion protein (121-231). The differential effect of macromolecular crowding as a function of protein topology suffices to make non-native configurations relatively more accessible.

Cytoskeletal Components Define Protein Location to Membrane Microdomains*

Science.gov (United States)

Szymanski, Witold G.; Zauber, Henrik; Erban, Alexander; Gorka, Michal; Wu, Xu Na; Schulze, Waltraud X.

2015-01-01

The plasma membrane is an important compartment that undergoes dynamic changes in composition upon external or internal stimuli. The dynamic subcompartmentation of proteins in ordered low-density (DRM) and disordered high-density (DSM) membrane phases is hypothesized to require interactions with cytoskeletal components. Here, we systematically analyzed the effects of actin or tubulin disruption on the distribution of proteins between membrane density phases. We used a proteomic screen to identify candidate proteins with altered submembrane location, followed by biochemical or cell biological characterization in Arabidopsis thaliana. We found that several proteins, such as plasma membrane ATPases, receptor kinases, or remorins resulted in a differential distribution between membrane density phases upon cytoskeletal disruption. Moreover, in most cases, contrasting effects were observed: Disruption of actin filaments largely led to a redistribution of proteins from DRM to DSM membrane fractions while disruption of tubulins resulted in general depletion of proteins from the membranes. We conclude that actin filaments are necessary for dynamic movement of proteins between different membrane phases and that microtubules are not necessarily important for formation of microdomains as such, but rather they may control the protein amount present in the membrane phases. PMID:26091700

Trophoblast cell fusion and differentiation are mediated by both the protein kinase C and a pathways.

Directory of Open Access Journals (Sweden)

Waka Omata

Full Text Available The syncytiotrophoblast of the human placenta is an epithelial barrier that interacts with maternal blood and is a key for the transfer of nutrients and other solutes to the developing fetus. The syncytiotrophoblast is a true syncytium and fusion of progenitor cytotrophoblasts is the cardinal event leading to the formation of this layer. BeWo cells are often used as a surrogate for cytotrophoblasts, since they can be induced to fuse, and then express certain differentiation markers associated with trophoblast syncytialization. Dysferlin, a syncytiotrophoblast membrane repair protein, is up-regulated in BeWo cells induced to fuse by treatment with forskolin; this fusion is thought to occur through cAMP/protein kinase A-dependent mechanisms. We hypothesized that dysferlin may also be up-regulated in response to fusion through other pathways. Here, we show that BeWo cells can also be induced to fuse by treatment with an activator of protein kinase C, and that this fusion is accompanied by increased expression of dysferlin. Moreover, a dramatic synergistic increase in dysferlin expression is observed when both the protein kinase A and protein kinase C pathways are activated in BeWo cells. This synergy in fusion is also accompanied by dramatic increases in mRNA for the placental fusion proteins syncytin 1, syncytin 2, as well as dysferlin. Dysferlin, however, was shown to be dispensable for stimulus-induced BeWo cell syncytialization, since dysferlin knockdown lines fused to the same extent as control cells. The classical trophoblast differentiation marker human chorionic gonadotropin was also monitored and changes in the expression closely parallel that of dysferlin in all of the experimental conditions employed. Thus different biochemical markers of trophoblast fusion behave in concert supporting the hypothesis that activation of both protein kinase C and A pathways lead to trophoblastic differentiation.

Phosphorylation site dynamics of early T-cell receptor signaling

DEFF Research Database (Denmark)

Chylek, Lily A; Akimov, Vyacheslav; Dengjel, Jörn

2014-01-01

In adaptive immune responses, T-cell receptor (TCR) signaling impacts multiple cellular processes and results in T-cell differentiation, proliferation, and cytokine production. Although individual protein-protein interactions and phosphorylation events have been studied extensively, we lack...... that diverse dynamic patterns emerge within seconds. We detected phosphorylation dynamics as early as 5 s and observed widespread regulation of key TCR signaling proteins by 30 s. Development of a computational model pointed to the presence of novel regulatory mechanisms controlling phosphorylation of sites...... a systems-level understanding of how these components cooperate to control signaling dynamics, especially during the crucial first seconds of stimulation. Here, we used quantitative proteomics to characterize reshaping of the T-cell phosphoproteome in response to TCR/CD28 co-stimulation, and found...

Differential protein expression in alligator leukocytes in response to bacterial lipopolysaccharide injection.

Science.gov (United States)

Merchant, Mark; Kinney, Clint; Sanders, Paige

2009-12-01

Blood was collected from three juvenile alligators (Alligator mississippiensis) before, and again 24h after, injection with bacterial lipopolysaccharide (LPS). The leukocytes were collected from both samples, and the proteins were extracted. Each group of proteins was labeled with a different fluorescent dye and the differences in protein expression were analyzed by two dimensional differential in-gel expressions (2D-DIGE). The proteins which appeared to be increased or decreased by treatment with LPS were selected and analyzed by MALDI-TOF to determine mass and LC-MS/MS to acquire the partial protein sequences. The peptide sequences were compared to the NCBI protein sequence database to determine homology with other sequences from other species. Several proteins of interest appeared to be increased upon LPS stimulation. Proteins with homology to human transgelin-2, fish glucose-6-phosphate dehydrogenase, amphibian α-enolase, alligator lactate dehydrogenase, fish ubiquitin-activating enzyme, and fungal β-tubulin were also increased after LPS injection. Proteins with homology to fish vimentin 4, murine heterogeneous nuclear ribonucleoprotein A3, and avian calreticulin were found to be decreased in response to LPS. In addition, five proteins, four of which were up-regulated (827, 560, 512, and 650%) and one that exhibited repressed expression (307%), did not show homology to any protein in the database, and thus may represent newly discovered proteins. We are using this biochemical approach to isolate and characterize alligator proteins with potential relevant immune function.

Plant GSK3 proteins regulate xylem cell differentiation downstream of TDIF-TDR signalling

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Kondo, Yuki; Ito, Tasuku; Nakagami, Hirofumi; Hirakawa, Yuki; Saito, Masato; Tamaki, Takayuki; Shirasu, Ken; Fukuda, Hiroo

2014-03-01

During plant radial growth typically seen in trees, procambial and cambial cells act as meristematic cells in the vascular system to self-proliferate and differentiate into xylem cells. These two processes are regulated by a signalling pathway composed of a peptide ligand and its receptor; tracheary element differentiation inhibitory factor (TDIF) and TDIF RECEPTOR (TDR). Here we show that glycogen synthase kinase 3 proteins (GSK3s) are crucial downstream components of the TDIF signalling pathway suppressing xylem differentiation from procambial cells. TDR interacts with GSK3s at the plasma membrane and activates GSK3s in a TDIF-dependent fashion. Consistently, a specific inhibitor of plant GSK3s strongly induces xylem cell differentiation through BRI1-EMS SUPPRESSOR 1 (BES1), a well-known target transcription factor of GSK3s. Our findings provide insight into the regulation of cell fate determination in meristem maintenance.

Differential Expression of Immunogenic Proteins on Virulent Mycobacterium tuberculosis Clinical Isolates

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Pablo Schierloh

2014-01-01

Full Text Available Molecular epidemiology has revealed that Mycobacterium tuberculosis (Mtb, formerly regarded as highly conserved species, displays a considerable degree of genetic variability that can influence the outcome of the disease as well as the innate and adaptive immune response. Recent studies have demonstrated that Mtb families found worldwide today differ in pathology, transmissibility, virulence, and development of immune response. By proteomic approaches seven proteins that were differentially expressed between a local clinical isolate from Latin-American-Mediterranean (LAM and from Haarlem (H lineages were identified. In order to analyze the immunogenic ability, recombinant Rv2241, Rv0009, Rv0407, and Rv2624c proteins were produced for testing specific antibody responses. We found that these proteins induced humoral immune responses in patients with drug-sensitive and drug-resistant tuberculosis with substantial cross-reactivity among the four proteins. Moreover, such reactivity was also correlated with anti-Mtb-cell surface IgM, but not with anti-ManLAM, anti-PPD, or anti-Mtb-surface IgG antibodies. Therefore, the present results describe new Mtb antigens with potential application as biomarkers of TB.

An Improved Differential Evolution Based Dynamic Economic Dispatch with Nonsmooth Fuel Cost Function

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R. Balamurugan

2007-09-01

Full Text Available Dynamic economic dispatch (DED is one of the major operational decisions in electric power systems. DED problem is an optimization problem with an objective to determine the optimal combination of power outputs for all generating units over a certain period of time in order to minimize the total fuel cost while satisfying dynamic operational constraints and load demand in each interval. This paper presents an improved differential evolution (IDE method to solve the DED problem of generating units considering valve-point effects. Heuristic crossover technique and gene swap operator are introduced in the proposed approach to improve the convergence characteristic of the differential evolution (DE algorithm. To illustrate the effectiveness of the proposed approach, two test systems consisting of five and ten generating units have been considered. The results obtained through the proposed method are compared with those reported in the literature.

Bone morphogenic protein-2 regulates the myogenic differentiation of PMVECs in CBDL rat serum-induced pulmonary microvascular remodeling

Energy Technology Data Exchange (ETDEWEB)

Liu, Chang; Chen, Lin; Zeng, Jing; Cui, Jian; Ning, Jiao-nin [Department of Anesthesia, Southwest Hospital, The Third Military Medical University, Chongqing 400038 (China); Wang, Guan-song [Institute of Respiratory Disease, Xinqiao Hospital, The Third Military Medical University, Chongqing 400037 (China); Belguise, Karine; Wang, Xiaobo [Université P. Sabatier Toulouse III and CNRS, LBCMCP, 31062 Toulouse Cedex 9 (France); Qian, Gui-sheng [Institute of Respiratory Disease, Xinqiao Hospital, The Third Military Medical University, Chongqing 400037 (China); Lu, Kai-zhi [Department of Anesthesia, Southwest Hospital, The Third Military Medical University, Chongqing 400038 (China); Yi, Bin, E-mail: yibin1974@163.com [Department of Anesthesia, Southwest Hospital, The Third Military Medical University, Chongqing 400038 (China)

2015-08-01

Hepatopulmonary syndrome (HPS) is characterized by an arterial oxygenation defect induced by intrapulmonary vasodilation (IPVD) that increases morbidity and mortality. In our previous study, it was determined that both the proliferation and the myogenic differentiation of pulmonary microvascular endothelial cells (PMVECs) play a key role in the development of IPVD. However, the molecular mechanism underlying the relationship between IPVD and the myogenic differentiation of PMVECs remains unknown. Additionally, it has been shown that bone morphogenic protein-2 (BMP2), via the control of protein expression, may regulate cell differentiation including cardiomyocyte differentiation, neuronal differentiation and odontoblastic differentiation. In this study, we observed that common bile duct ligation (CBDL)-rat serum induced the upregulation of the expression of several myogenic proteins (SM-α-actin, calponin, SM-MHC) and enhanced the expression levels of BMP2 mRNA and protein in PMVECs. We also observed that both the expression levels of Smad1/5 and the activation of phosphorylated Smad1/5 were significantly elevated in PMVECs following exposure to CBDL-rat serum, which was accompanied by the down-regulation of Smurf1. The blockage of the BMP2/Smad signaling pathway with Noggin inhibited the myogenic differentiation of PMVECs, a process that was associated with relatively low expression levels of both SM-α-actin and calponin in the setting of CBDL-rat serum exposure, although SM-MHC expression was not affected. These findings suggested that the BMP2/Smad signaling pathway is involved in the myogenic differentiation of the PMVECs. In conclusion, our data highlight the pivotal role of BMP2 in the CBDL-rat serum-induced myogenic differentiation of PMVECs via the activation of both Smad1 and Smad5 and the down-regulation of Smurf1, which may represent a potential therapy for HPS-induced pulmonary vascular remodeling. - Highlights: • CBDL-rat serum promotes the myogenic

Refinement of homology-based protein structures by molecular dynamics simulation techniques

NARCIS (Netherlands)

Fan, H; Mark, AE

The use of classical molecular dynamics simulations, performed in explicit water, for the refinement of structural models of proteins generated ab initio or based on homology has been investigated. The study involved a test set of 15 proteins that were previously used by Baker and coworkers to

Picosecond Fluorescence Dynamics of Tryptophan and 5-Fluorotryptophan in Monellin : Slow Water-Protein Relaxation Unmasked

NARCIS (Netherlands)

Xu, Jianhua; Chen, Binbin; Callis, Patrik Robert; Muiño, Pedro L; Rozeboom, Henriette J; Broos, Jaap; Toptygin, Dmitri; Brand, Ludwig; Knutson, Jay R

2015-01-01

Time Dependent Fluorescence Stokes (emission wavelength) Shifts (TDFSS) from tryptophan (Trp) following sub-picosecond excitation are increasingly used to investigate protein dynamics, most recently enabling active research interest into water dynamics near the surface of proteins. Unlike many

Dynamic nuclear polarization methods in solids and solutions to explore membrane proteins and membrane systems.

Science.gov (United States)

Cheng, Chi-Yuan; Han, Songi

2013-01-01

Membrane proteins regulate vital cellular processes, including signaling, ion transport, and vesicular trafficking. Obtaining experimental access to their structures, conformational fluctuations, orientations, locations, and hydration in membrane environments, as well as the lipid membrane properties, is critical to understanding their functions. Dynamic nuclear polarization (DNP) of frozen solids can dramatically boost the sensitivity of current solid-state nuclear magnetic resonance tools to enhance access to membrane protein structures in native membrane environments. Overhauser DNP in the solution state can map out the local and site-specific hydration dynamics landscape of membrane proteins and lipid membranes, critically complementing the structural and dynamics information obtained by electron paramagnetic resonance spectroscopy. Here, we provide an overview of how DNP methods in solids and solutions can significantly increase our understanding of membrane protein structures, dynamics, functions, and hydration in complex biological membrane environments.

Reverse micelles as a tool for probing solvent modulation of protein dynamics: Reverse micelle encapsulated hemoglobin☆

OpenAIRE

Roche, Camille J.; Dantsker, David; Heller, Elizabeth R.; Sabat, Joseph E.; Friedman, Joel M.

2013-01-01

Hydration waters impact protein dynamics. Dissecting the interplay between hydration waters and dynamics requires a protein that manifests a broad range of dynamics. Proteins in reverse micelles (RMs) have promise as tools to achieve this objective because the water content can be manipulated. Hemoglobin is an appropriate tool with which to probe hydration effects. We describe both a protocol for hemoglobin encapsulation in reverse micelles and a facile method using PEG and cosolvents to mani...

Natural loss-of-function mutation of myeloid differentiation protein 88 disrupts its ability to form Myddosomes

NARCIS (Netherlands)

Nagpal, K.; Plantinga, T.S.; Sirois, C.M.; Monks, B.G.; Latz, E.; Netea, M.G.; Golenbock, D.T.

2011-01-01

Myeloid differentiation protein 88 (MyD88) is a key signaling adapter in Toll-like receptor (TLR) signaling. MyD88 is also one of the most polymorphic adapter proteins. We screened the reported nonsynonymous coding mutations in MyD88 to identify variants with altered function. In reporter assays, a

Cellular prion protein is required for neuritogenesis: fine-tuning of multiple signaling pathways involved in focal adhesions and actin cytoskeleton dynamics

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Alleaume-Butaux A

2013-07-01

Full Text Available Aurélie Alleaume-Butaux,1,2 Caroline Dakowski,1,2 Mathéa Pietri,1,2 Sophie Mouillet-Richard,1,2 Jean-Marie Launay,3,4 Odile Kellermann,1,2 Benoit Schneider1,2 1INSERM, UMR-S 747, 2Paris Descartes University, Sorbonne Paris Cité, UMR-S 747, 3Public Hospital of Paris, Department of Biochemistry, INSERM UMR-S 942, Lariboisière Hospital, Paris, France; 4Pharma Research Department, Hoffmann La Roche Ltd, Basel, Switzerland Abstract: Neuritogenesis is a dynamic phenomenon associated with neuronal differentiation that allows a rather spherical neuronal stem cell to develop dendrites and axon, a prerequisite for the integration and transmission of signals. The acquisition of neuronal polarity occurs in three steps: (1 neurite sprouting, which consists of the formation of buds emerging from the postmitotic neuronal soma; (2 neurite outgrowth, which represents the conversion of buds into neurites, their elongation and evolution into axon or dendrites; and (3 the stability and plasticity of neuronal polarity. In neuronal stem cells, remodeling and activation of focal adhesions (FAs associated with deep modifications of the actin cytoskeleton is a prerequisite for neurite sprouting and subsequent neurite outgrowth. A multiple set of growth factors and interactors located in the extracellular matrix and the plasma membrane orchestrate neuritogenesis by acting on intracellular signaling effectors, notably small G proteins such as RhoA, Rac, and Cdc42, which are involved in actin turnover and the dynamics of FAs. The cellular prion protein (PrPC, a glycosylphosphatidylinositol (GPI-anchored membrane protein mainly known for its role in a group of fatal neurodegenerative diseases, has emerged as a central player in neuritogenesis. Here, we review the contribution of PrPC to neuronal polarization and detail the current knowledge on the signaling pathways fine-tuned by PrPC to promote neurite sprouting, outgrowth, and maintenance. We emphasize that Pr

Differential protein expression in mussels Mytilus galloprovincialis exposed to nano and ionic Ag

International Nuclear Information System (INIS)

Gomes, Tânia; Pereira, Catarina G.; Cardoso, Cátia; Bebianno, Maria João

2013-01-01

Highlights: •Different protein expression profiles between tissues and Ag forms. •Ag NPs and Ag + presented different mechanisms of toxic action. •Ag NPs toxicity is mediated by oxidative stress-induced cell signalling cascades. •New biomarkers for Ag NPs were proposed, i.e. MVP, ras partial and precol-P. -- Abstract: Ag NPs are one of the most commonly used NPs in nanotechnology whose environmental impacts are to date unknown and the information about bioavailability, mechanisms of biological uptake and toxic implications in organisms is scarce. So, the main objective of this study was to investigate differences in protein expression profiles in gills and digestive gland of mussels Mytilus galloprovincialis exposed to Ag NPs and Ag + (10 μg L −1 ) for a period of 15 days. Protein expression profiles of exposed gills and digestive glands were compared to those of control mussels using two–dimensional electrophoresis to discriminate differentially expressed proteins. Different patterns of protein expression were obtained for exposed mussels, dependent not only on the different redox requirements of each tissue but also to the Ag form used. Unique sets of differentially expressed proteins were affected by each silver form in addition to proteins that were affected by both Ag NPs and Ag + . Fifteen of these proteins were subsequently identified by MALDI–TOF–TOF and database search. Ag NPs affected similar cellular pathways as Ag + , with common response mechanisms in cytoskeleton and cell structure (catchin, myosin heavy chain), stress response (heat shock protein 70), oxidative stress (glutathione s-transferase), transcriptional regulation (nuclear receptor subfamily 1G), adhesion and mobility (precollagen-P) and energy metabolism (ATP synthase F0 subunit 6 and NADH dehydrogenase subunit 2). Exposure to Ag NPs altered the expression of two proteins associated with stress response (major vault protein and ras partial) and one protein involved in

Differential protein expression in mussels Mytilus galloprovincialis exposed to nano and ionic Ag

Energy Technology Data Exchange (ETDEWEB)

Gomes, Tânia; Pereira, Catarina G.; Cardoso, Cátia; Bebianno, Maria João, E-mail: mbebian@ualg.pt

2013-07-15

Highlights: •Different protein expression profiles between tissues and Ag forms. •Ag NPs and Ag{sup +} presented different mechanisms of toxic action. •Ag NPs toxicity is mediated by oxidative stress-induced cell signalling cascades. •New biomarkers for Ag NPs were proposed, i.e. MVP, ras partial and precol-P. -- Abstract: Ag NPs are one of the most commonly used NPs in nanotechnology whose environmental impacts are to date unknown and the information about bioavailability, mechanisms of biological uptake and toxic implications in organisms is scarce. So, the main objective of this study was to investigate differences in protein expression profiles in gills and digestive gland of mussels Mytilus galloprovincialis exposed to Ag NPs and Ag{sup +} (10 μg L{sup −1}) for a period of 15 days. Protein expression profiles of exposed gills and digestive glands were compared to those of control mussels using two–dimensional electrophoresis to discriminate differentially expressed proteins. Different patterns of protein expression were obtained for exposed mussels, dependent not only on the different redox requirements of each tissue but also to the Ag form used. Unique sets of differentially expressed proteins were affected by each silver form in addition to proteins that were affected by both Ag NPs and Ag{sup +}. Fifteen of these proteins were subsequently identified by MALDI–TOF–TOF and database search. Ag NPs affected similar cellular pathways as Ag{sup +}, with common response mechanisms in cytoskeleton and cell structure (catchin, myosin heavy chain), stress response (heat shock protein 70), oxidative stress (glutathione s-transferase), transcriptional regulation (nuclear receptor subfamily 1G), adhesion and mobility (precollagen-P) and energy metabolism (ATP synthase F0 subunit 6 and NADH dehydrogenase subunit 2). Exposure to Ag NPs altered the expression of two proteins associated with stress response (major vault protein and ras partial) and one

Normal mode analysis as a method to derive protein dynamics information from the Protein Data Bank.

Science.gov (United States)

Wako, Hiroshi; Endo, Shigeru

2017-12-01

Normal mode analysis (NMA) can facilitate quick and systematic investigation of protein dynamics using data from the Protein Data Bank (PDB). We developed an elastic network model-based NMA program using dihedral angles as independent variables. Compared to the NMA programs that use Cartesian coordinates as independent variables, key attributes of the proposed program are as follows: (1) chain connectivity related to the folding pattern of a polypeptide chain is naturally embedded in the model; (2) the full-atom system is acceptable, and owing to a considerably smaller number of independent variables, the PDB data can be used without further manipulation; (3) the number of variables can be easily reduced by some of the rotatable dihedral angles; (4) the PDB data for any molecule besides proteins can be considered without coarse-graining; and (5) individual motions of constituent subunits and ligand molecules can be easily decomposed into external and internal motions to examine their mutual and intrinsic motions. Its performance is illustrated with an example of a DNA-binding allosteric protein, a catabolite activator protein. In particular, the focus is on the conformational change upon cAMP and DNA binding, and on the communication between their binding sites remotely located from each other. In this illustration, NMA creates a vivid picture of the protein dynamics at various levels of the structures, i.e., atoms, residues, secondary structures, domains, subunits, and the complete system, including DNA and cAMP. Comparative studies of the specific protein in different states, e.g., apo- and holo-conformations, and free and complexed configurations, provide useful information for studying structurally and functionally important aspects of the protein.

Proteomic analysis of halotolerant proteins under high and low salt stress in Dunaliella salina using two-dimensional differential in-gel electrophoresis

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Yan-Long Jia

2016-01-01

Full Text Available Abstract Dunaliella salina, a single-celled marine alga with extreme salt tolerance, is an important model organism for studying fundamental extremophile survival mechanisms and their potential practical applications. In this study, two-dimensional differential in-gel electrophoresis (2D-DIGE was used to investigate the expression of halotolerant proteins under high (3 M NaCl and low (0.75 M NaCl salt concentrations. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS and bioinformatics were used to identify and characterize the differences among proteins. 2D-DIGE analysis revealed 141 protein spots that were significantly differentially expressed between the two salinities. Twenty-four differentially expressed protein spots were successfully identified by MALDI-TOF/TOF MS, including proteins in the following important categories: molecular chaperones, proteins involved in photosynthesis, proteins involved in respiration and proteins involved in amino acid synthesis. Expression levels of these proteins changed in response to the stress conditions, which suggests that they may be involved in the maintenance of intracellular osmotic pressure, cellular stress responses, physiological changes in metabolism, continuation of photosynthetic activity and other aspects of salt stress. The findings of this study enhance our understanding of the function and mechanisms of various proteins in salt stress.

Differential activation of G-proteins by μ-opioid receptor agonists

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Saidak, Zuzana; Blake-Palmer, Katherine; Hay, Debbie L; Northup, John K; Glass, Michelle

2006-01-01

We investigated the ability of the activated μ-opioid receptor (MOR) to differentiate between myristoylated Gαi1 and GαoA type Gα proteins, and the maximal activity of a range of synthetic and endogenous agonists to activate each Gα protein. Membranes from HEK293 cells stably expressing transfected MOR were chaotrope extracted to denature endogenous G-proteins and reconstituted with specific purified G-proteins. The Gα subunits were generated in bacteria and were demonstrated to be recognised equivalently to bovine brain purified Gα protein by CB1 cannabinoid receptors. The ability of agonists to catalyse the MOR-dependent GDP/[35S]GTPγS exchange was then compared for Gαi1 and GαoA. Activation of MOR by DAMGO produced a high-affinity saturable interaction for GαoA (Km=20±1 nM) but a low-affinity interaction with Gαi1 (Km=116±12 nM). DAMGO, met-enkephalin and leucine-enkephalin displayed maximal Gα activation among the agonists evaluated. Endomorphins 1 and 2, methadone and β-endorphin activated both Gα to more than 75% of the maximal response, whereas fentanyl partially activated both G-proteins. Buprenorphine and morphine demonstrated a statistically significant difference between the maximal activities between Gαi1 and GαoA. Interestingly, DAMGO, morphine, endomorphins 1 and 2, displayed significant differences in the potencies for the activation of the two Gα. Differences in maximal activity and potency, for Gαi1 versus GαoA, are both indicative of agonist selective activation of G-proteins in response to MOR activation. These findings may provide a starting point for the design of drugs that demonstrate greater selectivity between these two G-proteins and therefore produce a more limited range of effects. PMID:16415903

Dynamic changes in proteins during apple (Malus x domestica) fruit ripening and storage

OpenAIRE

Shi, Yun; Jiang, Li; Zhang, Li; Kang, Ruoyi; Yu, Zhifang

2014-01-01

A proteomic study, using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight, was conducted in apple fruit (cv. ‘Golden Delicious’) starting at 10 days prior to harvest through 50 days in storage. Total protein was extracted using a phenol/sodium dodecyl sulfate protocol. More than 400 protein spots were detected in each gel and 55 differentially expressed proteins (p

Proteomic profiling of human keratinocytes undergoing UVB-induced alternative differentiation reveals TRIpartite Motif Protein 29 as a survival factor.

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Véronique Bertrand-Vallery

Full Text Available BACKGROUND: Repeated exposures to UVB of human keratinocytes lacking functional p16(INK-4a and able to differentiate induce an alternative state of differentiation rather than stress-induced premature senescence. METHODOLOGY/PRINCIPAL FINDINGS: A 2D-DIGE proteomic profiling of this alternative state of differentiation was performed herein at various times after the exposures to UVB. Sixty-nine differentially abundant protein species were identified by mass spectrometry, many of which are involved in keratinocyte differentiation and survival. Among these protein species was TRIpartite Motif Protein 29 (TRIM29. Increased abundance of TRIM29 following UVB exposures was validated by Western blot using specific antibody and was also further analysed by immunochemistry and by RT-PCR. TRIM29 was found very abundant in keratinocytes and reconstructed epidermis. Knocking down the expression of TRIM29 by short-hairpin RNA interference decreased the viability of keratinocytes after UVB exposure. The abundance of involucrin mRNA, a marker of late differentiation, increased concomitantly. In TRIM29-knocked down reconstructed epidermis, the presence of picnotic cells revealed cell injury. Increased abundance of TRIM29 was also observed upon exposure to DNA damaging agents and PKC activation. The UVB-induced increase of TRIM29 abundance was dependent on a PKC signaling pathway, likely PKCdelta. CONCLUSIONS/SIGNIFICANCE: These findings suggest that TRIM29 allows keratinocytes to enter a protective alternative differentiation process rather than die massively after stress.

Organizing membrane-curving proteins: the emerging dynamical picture.

Science.gov (United States)

Simunovic, Mijo; Bassereau, Patricia; Voth, Gregory A

2018-03-30

Lipid membranes play key roles in cells, such as in trafficking, division, infection, remodeling of organelles, among others. The key step in all these processes is creating membrane curvature, typically under the control of many anchored, adhered or included proteins. However, it has become clear that the membrane itself can mediate the interactions among proteins to produce highly ordered assemblies. Computer simulations are ideally suited to investigate protein organization and the dynamics of membrane remodeling at near-micron scales, something that is extremely challenging to tackle experimentally. We review recent computational efforts in modeling protein-caused membrane deformation mechanisms, specifically focusing on coarse-grained simulations. We highlight work that exposed the membrane-mediated ordering of proteins into lines, meshwork, spirals and other assemblies, in what seems to be a very generic mechanism driven by a combination of short and long-ranged forces. Modulating the mechanical properties of membranes is an underexplored signaling mechanism in various processes deserving of more attention in the near future. Copyright © 2018 Elsevier Ltd. All rights reserved.

Differential representation of albumins and globulins during grain development in durum wheat and its possible functional consequences.

Science.gov (United States)

Arena, Simona; D'Ambrosio, Chiara; Vitale, Monica; Mazzeo, Fiorella; Mamone, Gianfranco; Di Stasio, Luigia; Maccaferri, Marco; Curci, Pasquale Luca; Sonnante, Gabriella; Zambrano, Nicola; Scaloni, Andrea

2017-06-06

Durum wheat (Triticum turgidum ssp. durum (Desf.) Husn.) is an economically important crop used for the production of semolina, which is the basis of pasta and other food products. Its grains provide proteins and starch for human consumption. Grain development is a key process in wheat physiology; it is highly affected by a number of enzymes that control the metabolic processes governing accumulation of starch and storage proteins and ultimately grain weight. Most of these enzymes are present in the albumin/globulin grain fraction, which represents about a quarter of total seed proteins. With the aim to describe the dynamic profile of the albumin/globulin fraction during durum wheat grain development, we performed a proteomic analysis of this subproteome using a two-dimensional differential gel electrophoresis (2D-DIGE)-based approach and compared six developmental stages. A total of 285 differentially (237 over- and 48 under-) represented spots was identified by nanoLC-ESI-LIT-MS/MS, which were associated with 217 non-redundant Triticum sequence entries. Quantitative protein dynamics demonstrated that carbon metabolism, energy, protein destination/storage, disease/defense and cell growth/division functional categories were highly affected during grain development, concomitantly with progressive grain size increase and starch/protein reserve accumulation. Bioinformatic interaction prediction revealed a complex network of differentially represented proteins mainly centered at enzymes involved in carbon and protein metabolism. A description of 18 proteins associated with wheat flour human allergies was also obtained; these components showed augmented levels at the last developmental stages. By providing a comprehensive understanding of the molecular basis of durum wheat grain development, yield and quality formation, this study provides the foundation and reveals potential biomarkers for further investigations of durum wheat breeding and semolina quality. A 2D

Finite-horizon differential games for missile-target interception system using adaptive dynamic programming with input constraints

Science.gov (United States)

Sun, Jingliang; Liu, Chunsheng

2018-01-01

In this paper, the problem of intercepting a manoeuvring target within a fixed final time is posed in a non-linear constrained zero-sum differential game framework. The Nash equilibrium solution is found by solving the finite-horizon constrained differential game problem via adaptive dynamic programming technique. Besides, a suitable non-quadratic functional is utilised to encode the control constraints into a differential game problem. The single critic network with constant weights and time-varying activation functions is constructed to approximate the solution of associated time-varying Hamilton-Jacobi-Isaacs equation online. To properly satisfy the terminal constraint, an additional error term is incorporated in a novel weight-updating law such that the terminal constraint error is also minimised over time. By utilising Lyapunov's direct method, the closed-loop differential game system and the estimation weight error of the critic network are proved to be uniformly ultimately bounded. Finally, the effectiveness of the proposed method is demonstrated by using a simple non-linear system and a non-linear missile-target interception system, assuming first-order dynamics for the interceptor and target.

Statistical Estimation of the Protein-Ligand Binding Free Energy Based On Direct Protein-Ligand Interaction Obtained by Molecular Dynamics Simulation

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Haruki Nakamura

2012-09-01

Full Text Available We have developed a method for estimating protein-ligand binding free energy (DG based on the direct protein-ligand interaction obtained by a molecular dynamics simulation. Using this method, we estimated the DG value statistically by the average values of the van der Waals and electrostatic interactions between each amino acid of the target protein and the ligand molecule. In addition, we introduced fluctuations in the accessible surface area (ASA and dihedral angles of the protein-ligand complex system as the entropy terms of the DG estimation. The present method included the fluctuation term of structural change of the protein and the effective dielectric constant. We applied this method to 34 protein-ligand complex structures. As a result, the correlation coefficient between the experimental and calculated DG values was 0.81, and the average error of DG was 1.2 kcal/mol with the use of the fixed parameters. These results were obtained from a 2 nsec molecular dynamics simulation.

Multiscale molecular dynamics simulations of membrane remodeling by Bin/Amphiphysin/Rvs family proteins

Science.gov (United States)

Chun, Chan; Haohua, Wen; Lanyuan, Lu; Jun, Fan

2016-01-01

Membrane curvature is no longer thought of as a passive property of the membrane; rather, it is considered as an active, regulated state that serves various purposes in the cell such as between cells and organelle definition. While transport is usually mediated by tiny membrane bubbles known as vesicles or membrane tubules, such communication requires complex interplay between the lipid bilayers and cytosolic proteins such as members of the Bin/Amphiphysin/Rvs (BAR) superfamily of proteins. With rapid developments in novel experimental techniques, membrane remodeling has become a rapidly emerging new field in recent years. Molecular dynamics (MD) simulations are important tools for obtaining atomistic information regarding the structural and dynamic aspects of biological systems and for understanding the physics-related aspects. The availability of more sophisticated experimental data poses challenges to the theoretical community for developing novel theoretical and computational techniques that can be used to better interpret the experimental results to obtain further functional insights. In this review, we summarize the general mechanisms underlying membrane remodeling controlled or mediated by proteins. While studies combining experiments and molecular dynamics simulations recall existing mechanistic models, concurrently, they extend the role of different BAR domain proteins during membrane remodeling processes. We review these recent findings, focusing on how multiscale molecular dynamics simulations aid in understanding the physical basis of BAR domain proteins, as a representative of membrane-remodeling proteins. Project supported by the National Natural Science Foundation of China (Grant No. 21403182) and the Research Grants Council of Hong Kong, China (Grant No. CityU 21300014).

Phosphoproteome analysis of streptomyces development reveals extensive protein phosphorylation accompanying bacterial differentiation

DEFF Research Database (Denmark)

Manteca, Angel; Ye, Juanying; Sánchez, Jesús

2011-01-01

Streptomycetes are bacterial species that undergo a complex developmental cycle that includes programmed cell death (PCD) events and sporulation. They are widely used in biotechnology because they produce most clinically relevant secondary metabolites. Although Streptomyces coelicolor is one...... events were detected during the presporulation and sporulation stages (80%). Most of these phosphorylations were not reported before in Streptomyces, and included sporulation factors, transcriptional regulators, protein kinases and other regulatory proteins. Several of the identified phosphorylated...... proteins, FtsZ, DivIVA, and FtsH2, were previously demonstrated to be involved in the sporulation process. We thus established for the first time the widespread occurrence and dynamic features of Ser/Thr/Tyr protein phosphorylation in a bacteria species and also revealed a previously unrecognized...

Receptor-interacting Protein 140 Overexpression Promotes Neuro-2a Neuronal Differentiation by ERK1/2 Signaling

Directory of Open Access Journals (Sweden)

Xiao Feng

2015-01-01

Full Text Available Background: Abnormal neuronal differentiation plays an important role in central nervous system (CNS development abnormalities such as Down syndrome (DS, a disorder that results directly from overexpression of genes in trisomic cells. Receptor-interacting protein 140 (RIP140 is significantly upregulated in DS brains, suggesting its involvement in DS CNS development abnormalities. However, the role of RIP140 in neuronal differentiation is still not clear. The current study aimed to investigate the effect of RIP140 overexpression on the differentiation of neuro-2a (N2a neuroblastoma cells, in vitro. Methods: Stably RIP140-overexpressing N2a (N2a-RIP140 cells were used as a neurodevelopmental model, and were constructed by lipofection and overexpression validated by real-time polymerase chain reaction and Western blot. Retinoic acid (RA was used to stimulate N2a differentiation. Combining the expression of Tuj1 at the mRNA and protein levels, the percentage of cells baring neurites, and the number of neurites per cell body was semi-quantified to determine the effect of RIP140 on differentiation of N2a cells. Furthermore, western blot and the ERK1/2 inhibitor U0126 were used to identify the specific signaling pathway by which RIP140 induces differentiation of N2a cells. Statistical significance of the differences between groups was determined by one-way analysis of variance followed by the Dunnett test. Results: Compared to untransfected N2a cells RIPl40 expression in N2a-RIP140 cells was remarkably upregulated at both the mRNA and protein levels. N2a-RIP140 cells had a significantly increased percentage of cells baring neurites, and numbers of neurites per cell, as compared to N2a cells, in the absence and presence of RA (P < 0.05. In addition, Tuj1, a neuronal biomarker, was strongly upregulated in N2a-RIP140 cells (P < 0.05 and phosphorylated ERK1/2 (p-ERK1/2 levels in N2a-RIP140 cells were dramatically increased, while differentiation was

Molecular dynamics simulations of protein-tyrosine phosphatase 1B. I. Ligand-induced changes in the protein motions

DEFF Research Database (Denmark)

Peters, Günther H. J.; Frimurer, T.M.; Andersen, J.N.

1999-01-01

Activity of enzymes, such as protein tyrosine phosphatases (PTPs), is often associated with structural changes in the enzyme, resulting in selective and stereospecific reactions with the substrate. To investigate the effect of a substrate on the motions occurring in PTPs, we have performed...... molecular dynamics simulations of PTP1B and PTP1B complexed with a high-affinity peptide DADEpYL, where pY stands for phosphorylated tyrosine. The peptide sequence is derived from the epidermal growth factor receptor (EGFR(988-993)). Simulations were performed in water for 1 ns, and the concerted motions...... in the protein were analyzed using the essential dynamics technique. Our results indicate that the predominately internal motions in PTP1B occur in a subspace of only a few degrees of freedom. Upon substrate binding, the flexibility of the protein is reduced by similar to 10%. The largest effect is found...

A Single-Cell Biochemistry Approach Reveals PAR Complex Dynamics during Cell Polarization.

Science.gov (United States)

Dickinson, Daniel J; Schwager, Francoise; Pintard, Lionel; Gotta, Monica; Goldstein, Bob

2017-08-21

Regulated protein-protein interactions are critical for cell signaling, differentiation, and development. For the study of dynamic regulation of protein interactions in vivo, there is a need for techniques that can yield time-resolved information and probe multiple protein binding partners simultaneously, using small amounts of starting material. Here we describe a single-cell protein interaction assay. Single-cell lysates are generated at defined time points and analyzed using single-molecule pull-down, yielding information about dynamic protein complex regulation in vivo. We established the utility of this approach by studying PAR polarity proteins, which mediate polarization of many animal cell types. We uncovered striking regulation of PAR complex composition and stoichiometry during Caenorhabditis elegans zygote polarization, which takes place in less than 20 min. PAR complex dynamics are linked to the cell cycle by Polo-like kinase 1 and govern the movement of PAR proteins to establish polarity. Our results demonstrate an approach to study dynamic biochemical events in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Study of the interaction of potassium ion channel protein with micelle by molecular dynamics simulation

Science.gov (United States)

Shantappa, Anil; Talukdar, Keka

2018-04-01

Ion channels are proteins forming pore inside the body of all living organisms. This potassium ion channel known as KcsA channel and it is found in the each cell and nervous system. Flow of various ions is regulated by the function of the ion channels. The nerve ion channel protein with protein data bank entry 1BL8, which is basically an ion channel protein in Streptomyces Lividans and which is taken up to form micelle-protein system and the system is analyzed by using molecular dynamics simulation. Firstly, ion channel pore is engineered by CHARMM potential and then Micelle-protein system is subjected to molecular dynamics simulation. For some specific micelle concentration, the protein unfolding is observed.

Kazakh therapy on differential protein expression of Achilles tendon healing in a 7-day postoperative rabbit model.

Science.gov (United States)

Nuerai, Shawutali; Ainuer, Jialili; Jiasharete, Jielile; Darebai, Redati; Kayrat, Aldyarhan; Tang, Bin; Jiangannur, Zheyiken; Bai, Jingping; Makabel, Bolat

2011-12-01

To compare the effect of cast immobilization with that of early Kiymil arkili emdew (Kazakh exercise therapy) on the post-operative healing of Achilles tendon rupture in rabbits, and to observe the influence of early Kiymil arkili emdew on the differentially expressed proteins in the healing tendon. Forty-five New Zealand white rabbits were randomly divided into three groups (Arm A: control group; Arm B: postoperative immobilization group; and Arm C: postoperative early Kiymil arkili emdew group). After tenotomy, the rabbits of the two experimental groups received microsurgery to repair the ruptured tendons, and then received either cast immobilization or early Kiymil arkili emdew treatment. Achilles tendon tissue samples were collected 7 days after the surgery, and two-dimensional gel electrophoresis and MALDI-TOF-MS technique were used to analyze differentially expressed proteins in the tendon tissue of the three Arms. A total of 462.67 +/- 11.59, 532.33 +/- 27.79, and 515.33 +/- 6.56 protein spots were detected by the two-dimensional polyacrylamide gels in the Achilles tendon samples of the rabbits in Arms A, B, and C, respectively. Nineteen differentially expressed protein spots were randomly selected from Arm C. Among them, 7 were unique, and 15 had five times higher abundance than those in Arm B. These included annexin A2, gelsolin isoforms and alpha-1 Type III collagen. It was confirmed by western blot that gelsolin isoform b, annexin A2, etc. had specific and incremental expression in Arm C. The self-protective instincts of humans were overlooked in the classical postoperative treatment for Achilles tendon rupture with cast immobilization. Kiymil arkili emdew induced the specific and incremental expression of proteins in the repaired Achilles tendon in the early healing stage in a rabbit model, compared with those treated with postoperative cast immobilization. These differentially expressed proteins may contribute to the healing of the Achilles tendon via

Simulation of Protein Structure, Dynamics and Function in Organic Media

National Research Council Canada - National Science Library

Daggett, Valerie

1998-01-01

The overall goal of our ONR-sponsored research is to pursue realistic molecular modeling strudies pertinnent to the related properties of protein stability, dynamics, structure, function, and folding in aqueous solution...

Protein Dynamics in Organic Media at Varying Water Activity Studied by Molecular Dynamics Simulation

DEFF Research Database (Denmark)

Wedberg, Nils Hejle Rasmus Ingemar; Abildskov, Jens; Peters, Günther H.J.

2012-01-01

In nonaqueous enzymology, control of enzyme hydration is commonly approached by fixing the thermodynamic water activity of the medium. In this work, we present a strategy for evaluating the water activity in molecular dynamics simulations of proteins in water/organic solvent mixtures. The method...... relies on determining the water content of the bulk phase and uses a combination of Kirkwood−Buff theory and free energy calculations to determine corresponding activity coefficients. We apply the method in a molecular dynamics study of Candida antarctica lipase B in pure water and the organic solvents...

A dynamic study of protein secretion and aggregation in the secretory pathway.

Science.gov (United States)

Mossuto, Maria Francesca; Sannino, Sara; Mazza, Davide; Fagioli, Claudio; Vitale, Milena; Yoboue, Edgar Djaha; Sitia, Roberto; Anelli, Tiziana

2014-01-01

Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC) is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC.

A dynamic study of protein secretion and aggregation in the secretory pathway.

Directory of Open Access Journals (Sweden)

Maria Francesca Mossuto

Full Text Available Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC.

Communication: Microsecond dynamics of the protein and water affect electron transfer in a bacterial bc1 complex

Science.gov (United States)

Martin, Daniel R.; Matyushov, Dmitry V.

2015-04-01

Cross-membrane electron transport between cofactors localized in proteins of mitochondrial respiration and bacterial photosynthesis is the source of all biological energy. The statistics and dynamics of nuclear fluctuations in these protein/membrane/water heterogeneous systems are critical for their energetic efficiency. The results of 13 μs of atomistic molecular dynamics simulations of the membrane-bound bc1 bacterial complex are analyzed here. The reaction is affected by a broad spectrum of nuclear modes, with the slowest dynamics in the range of time-scales ˜0.1-1.6 μs contributing half of the reaction reorganization energy. Two reorganization energies are required to describe protein electron transfer due to dynamical arrest of protein conformations on the observation window. This mechanistic distinction allows significant lowering of activation barriers for reactions in proteins.

Communication: Microsecond dynamics of the protein and water affect electron transfer in a bacterial bc1 complex

International Nuclear Information System (INIS)

Martin, Daniel R.; Matyushov, Dmitry V.

2015-01-01

Cross-membrane electron transport between cofactors localized in proteins of mitochondrial respiration and bacterial photosynthesis is the source of all biological energy. The statistics and dynamics of nuclear fluctuations in these protein/membrane/water heterogeneous systems are critical for their energetic efficiency. The results of 13 μs of atomistic molecular dynamics simulations of the membrane-bound bc 1 bacterial complex are analyzed here. The reaction is affected by a broad spectrum of nuclear modes, with the slowest dynamics in the range of time-scales ∼0.1-1.6 μs contributing half of the reaction reorganization energy. Two reorganization energies are required to describe protein electron transfer due to dynamical arrest of protein conformations on the observation window. This mechanistic distinction allows significant lowering of activation barriers for reactions in proteins

Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering--A Comparison between Wild-Type Protein and a Hinge Mutant.

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Saara Laulumaa

Full Text Available Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS. The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.

Combined Dynamic Light Scattering and Raman Spectroscopy Approach for Characterizing the Aggregation of Therapeutic Proteins

Directory of Open Access Journals (Sweden)

E. Neil Lewis

2014-12-01

Full Text Available Determination of the physicochemical properties of protein therapeutics and their aggregates is critical for developing formulations that enhance product efficacy, stability, safety and manufacturability. Analytical challenges are compounded for materials: (1 that are formulated at high concentration, (2 that are formulated with a variety of excipients, and (3 that are available only in small volumes. In this article, a new instrument is described that measures protein secondary and tertiary structure, as well as molecular size, over a range of concentrations and formulation conditions of low volume samples. Specifically, characterization of colloidal and conformational stability is obtained through a combination of two well-established analytical techniques: dynamic light scattering (DLS and Raman spectroscopy, respectively. As the data for these two analytical modalities are collected on the same sample at the same time, the technique enables direct correlation between them, in addition to the more straightforward benefit of minimizing sample usage by providing multiple analytical measurements on the same aliquot non-destructively. The ability to differentiate between unfolding and aggregation that the combination of these techniques provides enables insights into underlying protein aggregation mechanisms. The article will report on mechanistic insights for aggregation that have been obtained from the application of this technique to the characterization of lysozyme, which was evaluated as a function of concentration and pH.

Chaotic expression dynamics implies pluripotency: when theory and experiment meet

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Furusawa Chikara

2009-05-01

Full Text Available Abstract Background During normal development, cells undergo a unidirectional course of differentiation that progressively decreases the number of cell types they can potentially become. Pluripotent stem cells can differentiate into several types of cells, but terminally differentiated cells cannot differentiate any further. A fundamental problem in stem cell biology is the characterization of the difference in cellular states, e.g., gene expression profiles, between pluripotent stem cells and terminally differentiated cells. Presentation of the hypothesis To address the problem, we developed a dynamical systems model of cells with intracellular protein expression dynamics and interactions with each other. According to extensive simulations, cells with irregular (chaotic oscillations in gene expression dynamics have the potential to differentiate into other cell types. During development, such complex oscillations are lost successively, leading to a loss of pluripotency. These simulation results, together with recent single-cell-level measurements in stem cells, led us to the following hypothesis regarding pluripotency: Chaotic oscillation in the expression of some genes leads to cell pluripotency and affords cellular state heterogeneity, which is supported by itinerancy over quasi-stable states. Differentiation stabilizes these states, leading to a loss of pluripotency. Testing the hypothesis To test the hypothesis, it is crucial to measure the time course of gene expression levels at the single-cell level by fluorescence microscopy and fluorescence-activated cell sorting (FACS analysis. By analyzing the time series of single-cell-level expression data, one can distinguish whether the variation in protein expression level over time is due only to stochasticity in expression dynamics or originates from the chaotic dynamics inherent to cells, as our hypothesis predicts. By further analyzing the expression in differentiated cell types, one can

Conformational Rigidity and Protein Dynamics at Distinct Timescales Regulate PTP1B Activity and Allostery.

Science.gov (United States)

Choy, Meng S; Li, Yang; Machado, Luciana E S F; Kunze, Micha B A; Connors, Christopher R; Wei, Xingyu; Lindorff-Larsen, Kresten; Page, Rebecca; Peti, Wolfgang

2017-02-16

Protein function originates from a cooperation of structural rigidity, dynamics at different timescales, and allostery. However, how these three pillars of protein function are integrated is still only poorly understood. Here we show how these pillars are connected in Protein Tyrosine Phosphatase 1B (PTP1B), a drug target for diabetes and cancer that catalyzes the dephosphorylation of numerous substrates in essential signaling pathways. By combining new experimental and computational data on WT-PTP1B and ≥10 PTP1B variants in multiple states, we discovered a fundamental and evolutionarily conserved CH/π switch that is critical for positioning the catalytically important WPD loop. Furthermore, our data show that PTP1B uses conformational and dynamic allostery to regulate its activity. This shows that both conformational rigidity and dynamics are essential for controlling protein activity. This connection between rigidity and dynamics at different timescales is likely a hallmark of all enzyme function. Copyright © 2017 Elsevier Inc. All rights reserved.

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions.

Science.gov (United States)

Saha, Tanumoy; Rathmann, Isabel; Galic, Milos

2017-07-11

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

Identification of potential nuclear reprogramming and differentiation factors by a novel selection method for cloning chromatin-binding proteins

International Nuclear Information System (INIS)

Wang Liu; Zheng Aihua; Yi Ling; Xu Chongren; Ding Mingxiao; Deng Hongkui

2004-01-01

Nuclear reprogramming is critical for animal cloning and stem cell creation through nuclear transfer, which requires extensive remodeling of chromosomal architecture involving dramatic changes in chromatin-binding proteins. To understand the mechanism of nuclear reprogramming, it is critical to identify chromatin-binding factors specify the reprogramming process. In this report, we have developed a high-throughput selection method, based on T7 phage display and chromatin immunoprecipitation, to isolate chromatin-binding factors expressed in mouse embryonic stem cells using primary mouse embryonic fibroblast chromatin. Seven chromatin-binding proteins have been isolated by this method. We have also isolated several chromatin-binding proteins involved in hepatocyte differentiation. Our method provides a powerful tool to rapidly and selectively identify chromatin-binding proteins. The method can be used to study epigenetic modification of chromatin during nuclear reprogramming, cell differentiation, and transdifferentiation

Mycoplasma hyopneumoniae and Mycoplasma flocculare differential domains from orthologous surface proteins induce distinct cellular immune responses in mice.

Science.gov (United States)

Leal, Fernanda Munhoz Dos Anjos; Virginio, Veridiana Gomes; Martello, Carolina Lumertz; Paes, Jéssica Andrade; Borges, Thiago J; Jaeger, Natália; Bonorino, Cristina; Ferreira, Henrique Bunselmeyer

2016-07-15

Mycoplasma hyopneumoniae and Mycoplasma flocculare are two genetically close species found in the swine respiratory tract. Despite their similarities, while M. hyopneumoniae is the causative agent of porcine enzootic pneumonia, M. flocculare is a commensal bacterium. Genomic and transcriptional comparative analyses so far failed to explain the difference in pathogenicity between these two species. We then hypothesized that such difference might be, at least in part, explained by amino acid sequence and immunological or functional differences between ortholog surface proteins. In line with that, it was verified that approximately 85% of the ortholog surface proteins from M. hyopneumoniae 7448 and M. flocculare present one or more differential domains. To experimentally assess possible immunological implications of this kind of difference, the extracellular differential domains from one pair of orthologous surface proteins (MHP7448_0612, from M. hyopneumoniae, and MF_00357, from M. flocculare) were expressed in E. coli and used to immunize mice. The recombinant polypeptides (rMHP61267-169 and rMF35767-196, respectively) induced distinct cellular immune responses. While, rMHP61267-169 induced both Th1 and Th2 responses, rMF35767-196 induced just an early pro-inflammatory response. These results indicate that immunological properties determined by differential domains in orthologous surface protein might play a role in pathogenicity, contributing to elicit specific and differential immune responses against each species. Copyright © 2016 Elsevier B.V. All rights reserved.

Progression of 3D Protein Structure and Dynamics Measurements

Science.gov (United States)

Sato-Tomita, Ayana; Sekiguchi, Hiroshi; Sasaki, Yuji C.

2018-06-01

New measurement methodologies have begun to be proposed with the recent progress in the life sciences. Here, we introduce two new methodologies, X-ray fluorescence holography for protein structural analysis and diffracted X-ray tracking (DXT), to observe the dynamic behaviors of individual single molecules.

Combining protein sequence, structure, and dynamics: A novel approach for functional evolution analysis of PAS domain superfamily.

Science.gov (United States)

Dong, Zheng; Zhou, Hongyu; Tao, Peng

2018-02-01

PAS domains are widespread in archaea, bacteria, and eukaryota, and play important roles in various functions. In this study, we aim to explore functional evolutionary relationship among proteins in the PAS domain superfamily in view of the sequence-structure-dynamics-function relationship. We collected protein sequences and crystal structure data from RCSB Protein Data Bank of the PAS domain superfamily belonging to three biological functions (nucleotide binding, photoreceptor activity, and transferase activity). Protein sequences were aligned and then used to select sequence-conserved residues and build phylogenetic tree. Three-dimensional structure alignment was also applied to obtain structure-conserved residues. The protein dynamics were analyzed using elastic network model (ENM) and validated by molecular dynamics (MD) simulation. The result showed that the proteins with same function could be grouped by sequence similarity, and proteins in different functional groups displayed statistically significant difference in their vibrational patterns. Interestingly, in all three functional groups, conserved amino acid residues identified by sequence and structure conservation analysis generally have a lower fluctuation than other residues. In addition, the fluctuation of conserved residues in each biological function group was strongly correlated with the corresponding biological function. This research suggested a direct connection in which the protein sequences were related to various functions through structural dynamics. This is a new attempt to delineate functional evolution of proteins using the integrated information of sequence, structure, and dynamics. © 2017 The Protein Society.

Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

Directory of Open Access Journals (Sweden)

Juan A. González-Vera

2015-11-01

Full Text Available Probing the dynamic activities of protein kinases in real-time in living cells constitutes a major challenge that requires specific and sensitive tools tailored to meet the particular demands associated with cellular imaging. The development of genetically-encoded and synthetic fluorescent biosensors has provided means of monitoring protein kinase activities in a non-invasive fashion in their native cellular environment with high spatial and temporal resolution. Here, we review existing technologies to probe different dynamic features of protein kinases and discuss limitations where new developments are required to implement more performant tools, in particular with respect to infrared and near-infrared fluorescent probes and strategies which enable improved signal-to-noise ratio and controlled activation of probes.

Dynamics and energetics of the mammalian phosphatidylinositol transfer protein phospholipid exchange cycle

DEFF Research Database (Denmark)

Grabon, Aby; Orłowski, Adam; Tripathi, Ashutosh

2017-01-01

. However, the details of the PITP-mediated lipid exchange cycle remain entirely obscure. Here, all-atom molecular dynamics simulations of the mammalian StART-like PtdIns/phosphatidylcholine (PtdCho) transfer protein PITPα, both on membrane bilayers and in solvated systems, informed downstream biochemical...... analyses that tested key aspects of the hypotheses generated by the molecular dynamics simulations. These studies provided five key insights into the PITPα lipid exchange cycle: (i) interaction of PITPα with the membrane is spontaneous and mediated by four specific protein substructures; (ii) the ability......Phosphatidylinositol-transfer proteins (PITPs) regulate phosphoinositide signaling in eukaryotic cells. The defining feature of PITPs is their ability to exchange phosphatidylinositol (PtdIns) molecules between membranes, and this property is central to PITP-mediated regulation of lipid signaling...

The Role of Chromatin-Associated Proteins in Cancer

DEFF Research Database (Denmark)

Helin, Kristian; Minucci, Saverio

2017-01-01

The organization of the chromatin structure is essential for maintaining cell-type-specific gene expression and therefore for cell identity. This structure is highly dynamic and is regulated by a large number of chromatin-associated proteins that are required for normal development...... and differentiation. Recurrent somatic mutations have been found with high frequency in genes coding for chromatin-associated proteins in cancer, and several of these are required for cancer maintenance. In this review, we discuss recent advances in understanding the role of chromatin-associated proteins...

Two-dimensional gel human protein databases offer a systematic approach to the study of cell proliferation and differentiation

DEFF Research Database (Denmark)

Celis, julio E.; Gesser, Borbala; Dejgaard, Kurt

1989-01-01

Human cellular protein databases have been established using computer-analyzed 2D gel electrophoresis. These databases, which include information on various properties of proteins, offer a global approach to the study of regulation of cell proliferation and differentiation. Furthermore, thanks...

Two dimensional gel human protein databases offer a systematic approach to the study of cell proliferation and differentiation

DEFF Research Database (Denmark)

Celis, J E; Gesser, B; Dejgaard, K

1989-01-01

Human cellular protein databases have been established using computer-analyzed 2D gel electrophoresis. These databases, which include information on various properties of proteins, offer a global approach to the study of regulation of cell proliferation and differentiation. Furthermore, thanks to...

The Dynamic Programming Method of Stochastic Differential Game for Functional Forward-Backward Stochastic System

Directory of Open Access Journals (Sweden)

Shaolin Ji

2013-01-01

Full Text Available This paper is devoted to a stochastic differential game (SDG of decoupled functional forward-backward stochastic differential equation (FBSDE. For our SDG, the associated upper and lower value functions of the SDG are defined through the solution of controlled functional backward stochastic differential equations (BSDEs. Applying the Girsanov transformation method introduced by Buckdahn and Li (2008, the upper and the lower value functions are shown to be deterministic. We also generalize the Hamilton-Jacobi-Bellman-Isaacs (HJBI equations to the path-dependent ones. By establishing the dynamic programming principal (DPP, we derive that the upper and the lower value functions are the viscosity solutions of the corresponding upper and the lower path-dependent HJBI equations, respectively.

Proteomic analysis of differential protein expression of achilles tendon in a rabbit model by two-dimensional polyacrylamide gel electrophoresis at 21 days postoperation.

Science.gov (United States)

Jielile, Jiasharete; Jialili, Ainuer; Sabirhazi, Gulnur; Shawutali, Nuerai; Redati, Darebai; Chen, Jiangtao; Tang, Bin; Bai, Jingping; Aldyarhan, Kayrat

2011-10-01

Postoperative early kinesitherapy has been advocated as an optimal method for treating Achilles tendon rupture. However, an insight into the rationale of how early kinesitherapy contributes to healing of Achilles tendon remains to be achieved, and research in the area of proteomic analysis of Achilles tendon has so far been lacking. Forty-two rabbits were randomized into control group, immobilization group, and early motion group, and received postoperative cast immobilization and early motion treatments. Achilles tendon samples were prepared 21 days following microsurgery, and the proteins were separated with two-dimensional polyacrylamide gel electrophoresis. Differentially expressed proteins were first recognized by PDQuest software, and then identified using peptide mass fingerprinting, tandem mass spectrometry, and database searching. A total of 463  ±  12, 511  ±  39, and 513  ±  80 protein spots were successfully detected in the two-dimensional polyacrylamide gels for the Achilles tendon samples of rabbits in the control group, immobilization group, and early motion group, respectively. There were 15, 8, and 9 unique proteins in these three groups, respectively, and some differentially expressed proteins were also identified in each group. It was indicated that some of the differentially expressed proteins were involved in various metabolism pathways and may play an important role in healing of Achilles tendon rupture. Postoperative early kinesitherapy resulted in differentially expressed proteins in ruptured Achilles tendon compared with those treated with postoperative cast immobilization. These differentially expressed proteins may contribute to healing of Achilles tendon rupture through a mechanobiological mechanism due to the application of postoperative early kinesitherapy.

Differential proteomics analysis to identify proteins and pathways associated with male sterility of soybean using iTRAQ-based strategy.

Science.gov (United States)

Li, Jiajia; Ding, Xianlong; Han, Shaohuai; He, Tingting; Zhang, Hao; Yang, Longshu; Yang, Shouping; Gai, Junyi

2016-04-14

To further elucidate the molecular mechanism of cytoplasmic male sterility (CMS) in soybean, a differential proteomic analysis was completed between the CMS line NJCMS1A and its maintainer NJCMS1B using iTRAQ-based strategy. As a result, 180 differential abundance proteins (DAPs) were identified, of which, 60 were down-regulated and 120 were up-regulated in NJCMS1A compared with NJCMS1B. Bioinformatic analysis showed that 167 DAPs were annotated in 41 Gene Ontology functional groups, 106 DAPs were classified into 20 clusters of orthologous groups of protein categories, and 128 DAPs were enrichment in 53 KEGG pathways. Fifteen differential level proteins/genes with the same expression pattern were identified in the further conjoint analysis of DAPs and the previously reported differential expression genes. Moreover, multiple reaction monitoring test, qRT-PCR analysis and enzyme activity assay validated that the iTRAQ results were reliable. Based on functional analysis of DAPs, we concluded that male sterility in NJCMS1A might be related to insufficiencies in energy supply, unbalance of protein synthesis and degradation, disruption of flavonoid synthesis, programmed cell death, abnormalities of substance metabolism, etc. These results might facilitate our understanding of the molecular mechanisms behind CMS in soybean. Soybean is an important global crop that provides protein and oil. Heterosis is a significantly potential approach to increase the yield of soybean. Cytoplasmic male sterility (CMS) plays a vital role in the production of hybrid seeds. However, the genetic and molecular mechanisms of male sterility in soybean still need to be further elucidated. In the present paper, a differential proteomic analysis was carried out and the results showed that several key proteins involved in key pathways were associated with male sterility in soybean. This work provides a new insight to understand the genetic and molecular mechanisms underlying CMS in soybean

Effect of dynamic high pressure homogenization on the aggregation state of soy protein.

Science.gov (United States)

Keerati-U-Rai, Maneephan; Corredig, Milena

2009-05-13

Although soy proteins are often employed as functional ingredients in oil-water emulsions, very little is known about the aggregation state of the proteins in solution and whether any changes occur to soy protein dispersions during homogenization. The effect of dynamic high pressure homogenization on the aggregation state of the proteins was investigated using microdifferential scanning calorimetry and high performance size exclusion chromatography coupled with multiangle laser light scattering. Soy protein isolates as well as glycinin and beta-conglycinin fractions were prepared from defatted soy flakes and redispersed in 50 mM sodium phosphate buffer at pH 7.4. The dispersions were then subjected to homogenization at two different pressures, 26 and 65 MPa. The results demonstrated that dynamic high pressure homogenization causes changes in the supramolecular structure of the soy proteins. Both beta-conglycinin and glycinin samples had an increased temperature of denaturation after homogenization. The chromatographic elution profile showed a reduction in the aggregate concentration with homogenization pressure for beta-conglycinin and an increase in the size of the soluble aggregates for glycinin and soy protein isolate.

[Retrospective analysis of influence of differential protein intake on renal prognosis for progressive chronic kidney disease].

Science.gov (United States)

Dai, Wendi; Yin, Daoxin; Cui, Wenying; Liu, Wenhu

2014-01-28

To explore retrospectively the influence of differential protein intake on renal prognosis for progressive chronic kidney disease (CKD). A total of 159 chronic kidney disease patients at stages 2, 3 and 4 were enrolled and a questionnaire survey was conducted from January 2009 to July 2012. They were followed monthly and their clinical data collected, including primary disease, blood pressure, body mass index and adverse events. Laboratory tests were performed every 3 months, including biochemical parameters, protein-energy malnutrition (PEM), diet reviews and daily protein intake (DPI). A simplified MDRD formula was employed to evaluate the level of estimated glomerular filtration rate (eGFR). According to the level of DPI, they were divided into 3 groups of very low protein diet (VLPD): DPI ≤ 0.6 g · kg(-1) · d(-1), low-protein diet (LPD): DPI >0.6-protein diet (NPD): DPI ≥ 0.8 · g · kg(-1) · d(-1). Among them, 4 cases (2.50%) progressed to uremia stage and received renal replacement therapy, 2(1.25%) experienced rapid decline in renal function, 9(5.66%) were hospitalized from cardio-cerebral diseases and the 2-year kidney survival rate was 97.5%. At the end of study, among 9 patients of PEM, 2 subjects had a serum level of albumin under 32 g/L and another 7 with a BMI 0.05). Within a certain range, differential protein intake may not significantly affect the prognosis of kidney for progressive CKD patients.

Secreted Clusterin protein inhibits osteoblast differentiation of bone marrow mesenchymal stem cells by suppressing ERK1/2 signaling pathway.

Science.gov (United States)

Abdallah, Basem M; Alzahrani, Abdullah M; Kassem, Moustapha

2018-05-01

Secreted Clusterin (sCLU, also known as Apolipoprotein J) is an anti-apoptotic glycoprotein involved in the regulation of cell proliferation, lipid transport, extracellular tissue remodeling and apoptosis. sCLU is expressed and secreted by mouse bone marrow-derived skeletal (stromal or mesenchymal) stem cells (mBMSCs), but its functional role in MSC biology is not known. In this study, we demonstrated that Clusterin mRNA expression and protein secretion in conditioned medium increased during adipocyte differentiation and decreased during osteoblast differentiation of mBMSCs. Treatment of mBMSC cultures with recombinant sCLU protein increased cell proliferation and exerted an inhibitory effect on the osteoblast differentiation while stimulated adipocyte differentiation in a dose-dependent manner. siRNA-mediated silencing of Clu expression in mBMSCs reduced adipocyte differentiation and stimulated osteoblast differentiation of mBMSCs. Furthermore, the inhibitory effect of sCLU on the osteoblast differentiation of mBMSCs was mediated by the suppression of extracellular signal-regulated kinase (ERK1/2) phosphorylation. In conclusion, we identified sCLU as a regulator of mBMSCs lineage commitment to osteoblasts versus adipocytes through a mechanism mediated by ERK1/2 signaling. Inhibiting sCLU is a possible therapeutic approach for enhancing osteoblast differentiation and consequently bone formation. Copyright © 2018 Elsevier Inc. All rights reserved.

Differential diagnosis of feline leukemia virus subgroups using pseudotype viruses expressing green fluorescent protein.

Science.gov (United States)

Nakamura, Megumi; Sato, Eiji; Miura, Tomoyuki; Baba, Kenji; Shimoda, Tetsuya; Miyazawa, Takayuki

2010-06-01

Feline leukemia virus (FeLV) is classified into three receptor interference subgroups, A, B and C. In this study, to differentiate FeLV subgroups, we developed a simple assay system using pseudotype viruses expressing green fluorescent protein (GFP). We prepared gfp pseudotype viruses, named gfp(FeLV-A), gfp(FeLV-B) and gfp(FeLV-C) harboring envelopes of FeLV-A, B and C, respectively. The gfp pseudotype viruses completely interfered with the same subgroups of FeLV reference strains on FEA cells (a feline embryonic fibroblast cell line). We also confirmed that the pseudotype viruses could differentiate FeLV subgroups in field isolates. The assay will be useful for differential diagnosis of FeLV subgroups in veterinary diagnostic laboratories in the future.

Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

KAUST Repository

Guttery, David  S.; Poulin, Benoit; Ramaprasad, Abhinay; Wall, Richard  J.; Ferguson, David  J.P.; Brady, Declan; Patzewitz, Eva-Maria; Whipple, Sarah; Straschil, Ursula; Wright, Megan  H.; Mohamed, Alyaa  M.A.H.; Radhakrishnan, Anand; Arold, Stefan T.; Tate, Edward  W.; Holder, Anthony  A.; Wickstead, Bill; Pain, Arnab; Tewari, Rita

2014-01-01

Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

KAUST Repository

Guttery, David S.

2014-07-09

Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

Palmitoylation regulates epidermal homeostasis and hair follicle differentiation.

Directory of Open Access Journals (Sweden)

Pleasantine Mill

2009-11-01

Full Text Available Palmitoylation is a key post-translational modification mediated by a family of DHHC-containing palmitoyl acyl-transferases (PATs. Unlike other lipid modifications, palmitoylation is reversible and thus often regulates dynamic protein interactions. We find that the mouse hair loss mutant, depilated, (dep is due to a single amino acid deletion in the PAT, Zdhhc21, resulting in protein mislocalization and loss of palmitoylation activity. We examined expression of Zdhhc21 protein in skin and find it restricted to specific hair lineages. Loss of Zdhhc21 function results in delayed hair shaft differentiation, at the site of expression of the gene, but also leads to hyperplasia of the interfollicular epidermis (IFE and sebaceous glands, distant from the expression site. The specific delay in follicle differentiation is associated with attenuated anagen propagation and is reflected by decreased levels of Lef1, nuclear beta-catenin, and Foxn1 in hair shaft progenitors. In the thickened basal compartment of mutant IFE, phospho-ERK and cell proliferation are increased, suggesting increased signaling through EGFR or integrin-related receptors, with a parallel reduction in expression of the key differentiation factor Gata3. We show that the Src-family kinase, Fyn, involved in keratinocyte differentiation, is a direct palmitoylation target of Zdhhc21 and is mislocalized in mutant follicles. This study is the first to demonstrate a key role for palmitoylation in regulating developmental signals in mammalian tissue homeostasis.

Differentially expressed and survival-related proteins of lung adenocarcinoma with bone metastasis.

Science.gov (United States)

Yang, Mengdi; Sun, Yi; Sun, Jing; Wang, Zhiyu; Zhou, Yiyi; Yao, Guangyu; Gu, Yifeng; Zhang, Huizhen; Zhao, Hui

2018-04-01

Despite recent advances in targeted and immune-based therapies, the poor prognosis of lung adenocarcinoma (LUAD) with bone metastasis (BM) remains a challenge. First, two-dimensional gel electrophoresis (2-DE) was used to identify proteins that were differentially expressed in LUAD with BM, and then matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) was used to identify these proteins. Second, the Cancer Genome Atlas (TCGA) was used to identify mutations in these differentially expressed proteins and Kaplan-Meier plotter (KM Plotter) was used to generate survival curves for the analyzed cases. Immunohistochemistry (IHC) was used to check the expression of proteins in 28 patients with BM and nine patients with LUAD. Lastly, the results were analyzed with respect to clinical features and patient's follow-up. We identified a number of matched proteins from 2-DE. High expression of enolase 1 (ENO1) (HR = 1.67, logrank P = 1.9E-05), ribosomal protein lateral stalk subunit P2 (RPLP2) (HR = 1.77, logrank P = 2.9e-06), and NME/NM23 nucleoside diphosphate kinase 2 (NME1-NME2) (HR = 2.65, logrank P = 3.9E-15) was all significantly associated with poor survival (P < 0.05). Further, ENO1 was upregulated (P = 0.0004) and calcyphosine (CAPS1) was downregulated (P = 5.34E-07) in TCGA LUAD RNA-seq expression data. IHC revealed that prominent ENO1 staining (OR = 7.5, P = 0.034) and low levels of CAPS1 (OR = 0.01, P < 0.0001) staining were associated with BM incidence. Finally, we found that LUAD patients with high expression of ENO1 and RPLP2 had worse overall survival. This is the first instance where the genes ENO1, RPLP2, NME1-NME2 and CAPS1 were associated with disease severity and progression in LUAD patients with BM. Thus, with this study, we have identified potential biomarkers and therapeutic targets for this disease. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Unveiling protein functions through the dynamics of the interaction network.

Directory of Open Access Journals (Sweden)

Irene Sendiña-Nadal

Full Text Available Protein interaction networks have become a tool to study biological processes, either for predicting molecular functions or for designing proper new drugs to regulate the main biological interactions. Furthermore, such networks are known to be organized in sub-networks of proteins contributing to the same cellular function. However, the protein function prediction is not accurate and each protein has traditionally been assigned to only one function by the network formalism. By considering the network of the physical interactions between proteins of the yeast together with a manual and single functional classification scheme, we introduce a method able to reveal important information on protein function, at both micro- and macro-scale. In particular, the inspection of the properties of oscillatory dynamics on top of the protein interaction network leads to the identification of misclassification problems in protein function assignments, as well as to unveil correct identification of protein functions. We also demonstrate that our approach can give a network representation of the meta-organization of biological processes by unraveling the interactions between different functional classes.

Differential saliva-induced breakdown of starch filled protein gels in relation to sensory perception

NARCIS (Netherlands)

Janssen, A.M.; Pijpekamp, A.M. van de; Labiausse, D.

2009-01-01

In this study, the differential breakdown of protein gels containing four types of high and low cross-linked starch granules were studied. Susceptibility to saliva-induced breakdown of starch granules and the consequences of these for overall breakdown of the gel matrix were captured using a

Protein complexes and cholesterol in the control of late endosomal dynamicsCholesterol and multi-protein complexes in the control of late endosomal dynamics

NARCIS (Netherlands)

Kant, Rik Henricus Nicolaas van der

2013-01-01

Late endosomal transport is disrupted in several diseases such as Niemann-Pick type C, ARC syndrome and Alzheimer’s disease. This thesis describes the regulation of late endosomal dynamics by cholesterol and multi-protein complexes. We find that cholesterol acts as a cellular tomtom that steers the

Extending Differential Fault Analysis to Dynamic S-Box Advanced Encryption Standard Implementations

Science.gov (United States)

2014-09-18

number. As a result decryption is a different function which relies on a different key to efficiently undo the work of encryption . RSA is the most...EXTENDING DIFFERENTIAL FAULT ANALYSIS TO DYNAMIC S-BOX ADVANCED ENCRYPTION STANDARD IMPLEMENTATIONS THESIS Bradley M. Flamm, Civilian AFIT-ENG-T-14-S...ADVANCED ENCRYPTION STANDARD IMPLEMENTATIONS THESIS Presented to the Faculty Department of Electrical and Computer Engineering Graduate School of

Zaccai neutron resilience and site-specific hydration dynamics in a globular protein

Energy Technology Data Exchange (ETDEWEB)

Miao, Yinglong [Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Hong, Liang [Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Yi, Zheng [Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Smith, Jeremy C. [Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

2013-07-16

A discussion is presented of contributions of the Zaccai group to the understanding of flexibility in biological macromolecules using dynamic neutron scattering. The concept of resilience as introduced by Zaccai is discussed and investigated using molecular dynamics simulation on camphor-bound cytochrome P450. The resilience of hydrophilic residues is found to be more strongly affected by hydration than that of hydrophobic counterparts. The hydration-induced softening of protein propagates from the surface into the dry core. Furthermore, buried hydrophilic residues behave more like those exposed on the protein surface, and are different from their hydrophobic counterparts.

Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy.

Science.gov (United States)

Delaforge, Elise; Kragelj, Jaka; Tengo, Laura; Palencia, Andrés; Milles, Sigrid; Bouvignies, Guillaume; Salvi, Nicola; Blackledge, Martin; Jensen, Malene Ringkjøbing

2018-01-24

Intrinsically disordered proteins (IDPs) display a large number of interaction modes including folding-upon-binding, binding without major structural transitions, or binding through highly dynamic, so-called fuzzy, complexes. The vast majority of experimental information about IDP binding modes have been inferred from crystal structures of proteins in complex with short peptides of IDPs. However, crystal structures provide a mainly static view of the complexes and do not give information about the conformational dynamics experienced by the IDP in the bound state. Knowledge of the dynamics of IDP complexes is of fundamental importance to understand how IDPs engage in highly specific interactions without concomitantly high binding affinity. Here, we combine rotating-frame R 1ρ , Carr-Purcell-Meiboom Gill relaxation dispersion as well as chemical exchange saturation transfer to decipher the dynamic interaction profile of an IDP in complex with its partner. We apply the approach to the dynamic signaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disordered regulatory domain of the MAPK kinase MKK4. Our study demonstrates that MKK4 employs a subtle combination of interaction modes in order to bind to p38α, leading to a complex displaying significantly different dynamics across the bound regions.

Extracting protein dynamics information from overlapped NMR signals using relaxation dispersion difference NMR spectroscopy.

Science.gov (United States)

Konuma, Tsuyoshi; Harada, Erisa; Sugase, Kenji

2015-12-01

Protein dynamics plays important roles in many biological events, such as ligand binding and enzyme reactions. NMR is mostly used for investigating such protein dynamics in a site-specific manner. Recently, NMR has been actively applied to large proteins and intrinsically disordered proteins, which are attractive research targets. However, signal overlap, which is often observed for such proteins, hampers accurate analysis of NMR data. In this study, we have developed a new methodology called relaxation dispersion difference that can extract conformational exchange parameters from overlapped NMR signals measured using relaxation dispersion spectroscopy. In relaxation dispersion measurements, the signal intensities of fluctuating residues vary according to the Carr-Purcell-Meiboon-Gill pulsing interval, whereas those of non-fluctuating residues are constant. Therefore, subtraction of each relaxation dispersion spectrum from that with the highest signal intensities, measured at the shortest pulsing interval, leaves only the signals of the fluctuating residues. This is the principle of the relaxation dispersion difference method. This new method enabled us to extract exchange parameters from overlapped signals of heme oxygenase-1, which is a relatively large protein. The results indicate that the structural flexibility of a kink in the heme-binding site is important for efficient heme binding. Relaxation dispersion difference requires neither selectively labeled samples nor modification of pulse programs; thus it will have wide applications in protein dynamics analysis.

Extracting protein dynamics information from overlapped NMR signals using relaxation dispersion difference NMR spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Konuma, Tsuyoshi [Icahn School of Medicine at Mount Sinai, Department of Structural and Chemical Biology (United States); Harada, Erisa [Suntory Foundation for Life Sciences, Bioorganic Research Institute (Japan); Sugase, Kenji, E-mail: sugase@sunbor.or.jp, E-mail: sugase@moleng.kyoto-u.ac.jp [Kyoto University, Department of Molecular Engineering, Graduate School of Engineering (Japan)

2015-12-15

Protein dynamics plays important roles in many biological events, such as ligand binding and enzyme reactions. NMR is mostly used for investigating such protein dynamics in a site-specific manner. Recently, NMR has been actively applied to large proteins and intrinsically disordered proteins, which are attractive research targets. However, signal overlap, which is often observed for such proteins, hampers accurate analysis of NMR data. In this study, we have developed a new methodology called relaxation dispersion difference that can extract conformational exchange parameters from overlapped NMR signals measured using relaxation dispersion spectroscopy. In relaxation dispersion measurements, the signal intensities of fluctuating residues vary according to the Carr-Purcell-Meiboon-Gill pulsing interval, whereas those of non-fluctuating residues are constant. Therefore, subtraction of each relaxation dispersion spectrum from that with the highest signal intensities, measured at the shortest pulsing interval, leaves only the signals of the fluctuating residues. This is the principle of the relaxation dispersion difference method. This new method enabled us to extract exchange parameters from overlapped signals of heme oxygenase-1, which is a relatively large protein. The results indicate that the structural flexibility of a kink in the heme-binding site is important for efficient heme binding. Relaxation dispersion difference requires neither selectively labeled samples nor modification of pulse programs; thus it will have wide applications in protein dynamics analysis.

The consistency of large concerted motions in proteins in molecular dynamics simulations

NARCIS (Netherlands)

de Groot, B.L.; van Aalten, D.M.F.; Amadei, A; Berendsen, H.J.C.

1996-01-01

A detailed investigation is presented into the effect of limited sampling time and small changes in the force field on molecular dynamics simulations of a protein. Thirteen independent simulations of the B1 IgG-binding domain of streptococcal protein G were performed, with small changes in the

Differential Neural Networks for Identification and Filtering in Nonlinear Dynamic Games

Directory of Open Access Journals (Sweden)

Emmanuel García

2014-01-01

Full Text Available This paper deals with the problem of identifying and filtering a class of continuous-time nonlinear dynamic games (nonlinear differential games subject to additive and undesired deterministic perturbations. Moreover, the mathematical model of this class is completely unknown with the exception of the control actions of each player, and even though the deterministic noises are known, their power (or their effect is not. Therefore, two differential neural networks are designed in order to obtain a feedback (perfect state information pattern for the mentioned class of games. In this way, the stability conditions for two state identification errors and for a filtering error are established, the upper bounds of these errors are obtained, and two new learning laws for each neural network are suggested. Finally, an illustrating example shows the applicability of this approach.

Dynamics of a Bertrand duopoly with differentiated products and nonlinear costs: Analysis, comparisons and new evidences

International Nuclear Information System (INIS)

Brianzoni, Serena; Gori, Luca; Michetti, Elisabetta

2015-01-01

This paper studies mathematical properties and dynamics of a duopoly with price competition and horizontal product differentiation by introducing quadratic production costs (decreasing returns to scale), thus extending the model with linear costs (constant returns to scale) of Fanti et al. [11]. The economy is described by a two-dimensional non-invertible discrete time dynamic system. The paper first determines fixed points and other invariant sets, showing that synchronized dynamics can occur. Then, stability properties are compared in the cases of quadratic costs and linear costs by considering the degree of product differentiation and the speed of adjustment of prices as key parameters. It is also shown that synchronization takes place if products tend to be relatively complements and stressed similarities and differences between models with quadratic and linear costs. Finally, the paper focuses on the phenomenon of multistability thus underlying new evidences in comparison with the model with linear costs.

Dynamic changes in protein functional linkage networks revealed by integration with gene expression data.

Directory of Open Access Journals (Sweden)

Shubhada R Hegde

2008-11-01

Full Text Available Response of cells to changing environmental conditions is governed by the dynamics of intricate biomolecular interactions. It may be reasonable to assume, proteins being the dominant macromolecules that carry out routine cellular functions, that understanding the dynamics of protein:protein interactions might yield useful insights into the cellular responses. The large-scale protein interaction data sets are, however, unable to capture the changes in the profile of protein:protein interactions. In order to understand how these interactions change dynamically, we have constructed conditional protein linkages for Escherichia coli by integrating functional linkages and gene expression information. As a case study, we have chosen to analyze UV exposure in wild-type and SOS deficient E. coli at 20 minutes post irradiation. The conditional networks exhibit similar topological properties. Although the global topological properties of the networks are similar, many subtle local changes are observed, which are suggestive of the cellular response to the perturbations. Some such changes correspond to differences in the path lengths among the nodes of carbohydrate metabolism correlating with its loss in efficiency in the UV treated cells. Similarly, expression of hubs under unique conditions reflects the importance of these genes. Various centrality measures applied to the networks indicate increased importance for replication, repair, and other stress proteins for the cells under UV treatment, as anticipated. We thus propose a novel approach for studying an organism at the systems level by integrating genome-wide functional linkages and the gene expression data.

Correlation of chemical shifts predicted by molecular dynamics simulations for partially disordered proteins

Energy Technology Data Exchange (ETDEWEB)

Karp, Jerome M.; Erylimaz, Ertan; Cowburn, David, E-mail: cowburn@cowburnlab.org, E-mail: David.cowburn@einstein.yu.edu [Albert Einstein College of Medicine of Yeshiva University, Department of Biochemistry (United States)

2015-01-15

There has been a longstanding interest in being able to accurately predict NMR chemical shifts from structural data. Recent studies have focused on using molecular dynamics (MD) simulation data as input for improved prediction. Here we examine the accuracy of chemical shift prediction for intein systems, which have regions of intrinsic disorder. We find that using MD simulation data as input for chemical shift prediction does not consistently improve prediction accuracy over use of a static X-ray crystal structure. This appears to result from the complex conformational ensemble of the disordered protein segments. We show that using accelerated molecular dynamics (aMD) simulations improves chemical shift prediction, suggesting that methods which better sample the conformational ensemble like aMD are more appropriate tools for use in chemical shift prediction for proteins with disordered regions. Moreover, our study suggests that data accurately reflecting protein dynamics must be used as input for chemical shift prediction in order to correctly predict chemical shifts in systems with disorder.

Induction of DNA damage in γ-irradiated nuclei stripped of nuclear protein classes: differential modulation of double-strand break and DNA-protein crosslink formation

International Nuclear Information System (INIS)

Xue, L.-Y.; Friedman, L.R.; Oleinick, N.L.; Chiu, S.-M.

1994-01-01

The influence of chromatin proteins on the induction of DNA double-strand breaks (dsb) and DNA-protein crosslinks (dpc) by γ-radiation was investigated. Low molecular weight non-histone proteins and classes of histones were extracted with increasing concentrations of NaC1, whereas nuclear matrix proteins were not extractable even by 2.0 M NACl. The yield of dsb increased with progressive removal of proteins from chromatin. The data support our previous conclusion that nuclear matrix protein rather than the majority of the histones are the predominant substrates for dpc production, although the involvement of a subset of tightly bound histones (H3 and H4) has not been excluded. This finding demonstrates that chromatin proteins can differentially modify the yield of two types of radiation-induced DNA lesions. (author)

Communication: Microsecond dynamics of the protein and water affect electron transfer in a bacterial bc{sub 1} complex

Energy Technology Data Exchange (ETDEWEB)

Martin, Daniel R.; Matyushov, Dmitry V., E-mail: dmitrym@asu.edu [Department of Physics and Department of Chemistry and Biochemistry, Arizona State University, P.O. Box 871504, Tempe, Arizona 85287 (United States)

2015-04-28

Cross-membrane electron transport between cofactors localized in proteins of mitochondrial respiration and bacterial photosynthesis is the source of all biological energy. The statistics and dynamics of nuclear fluctuations in these protein/membrane/water heterogeneous systems are critical for their energetic efficiency. The results of 13 μs of atomistic molecular dynamics simulations of the membrane-bound bc{sub 1} bacterial complex are analyzed here. The reaction is affected by a broad spectrum of nuclear modes, with the slowest dynamics in the range of time-scales ∼0.1-1.6 μs contributing half of the reaction reorganization energy. Two reorganization energies are required to describe protein electron transfer due to dynamical arrest of protein conformations on the observation window. This mechanistic distinction allows significant lowering of activation barriers for reactions in proteins.

Synthesis of mitochondrial uncoupling protein in brown adipocytes differentiated in cell culture

International Nuclear Information System (INIS)

Kopecky, J.; Baudysova, M.; Zanotti, F.; Janikova, D.; Pavelka, S.; Houstek, J.

1990-01-01

In order to characterize the biogenesis of unique thermogenic mitochondria of brown adipose tissue, differentiation of precursor cells isolated from mouse brown adipose tissue was studied in cell culture. Synthesis of mitochondrial uncoupling protein (UCP), F1-ATPase, and cytochrome oxidase was examined by L-[35S]methionine labeling and immunoblotting. For the first time, synthesis of physiological amounts of the UCP, a key and tissue-specific component of thermogenic mitochondria, was observed in cultures at about confluence (day 6), indicating that a complete differentiation of brown adipocytes was achieved in vitro. In postconfluent cells (day 8) the content of UCP decreased rapidly, in contrast to some other mitochondrial proteins (beta subunit of F1-ATPase, cytochrome oxidase). In these cells, it was possible, by using norepinephrine, to induce specifically the synthesis of the UCP but not of F1-ATPase or cytochrome oxidase. The maximal response was observed at 0.1 microM norepinephrine and the synthesis of UCP remained activated for at least 24 h. Detailed analysis revealed a major role of the beta-adrenergic receptors and elevated intracellular concentration of cAMP in stimulation of UCP synthesis. A quantitative recovery of the newly synthesized UCP in the mitochondrial fraction indicated completed biogenesis of functionally competent thermogenic mitochondria

Conformational dynamics of ATP/Mg:ATP in motor proteins via data mining and molecular simulation

Science.gov (United States)

Bojovschi, A.; Liu, Ming S.; Sadus, Richard J.

2012-08-01

The conformational diversity of ATP/Mg:ATP in motor proteins was investigated using molecular dynamics and data mining. Adenosine triphosphate (ATP) conformations were found to be constrained mostly by inter cavity motifs in the motor proteins. It is demonstrated that ATP favors extended conformations in the tight pockets of motor proteins such as F1-ATPase and actin whereas compact structures are favored in motor proteins such as RNA polymerase and DNA helicase. The incorporation of Mg2+ leads to increased flexibility of ATP molecules. The differences in the conformational dynamics of ATP/Mg:ATP in various motor proteins was quantified by the radius of gyration. The relationship between the simulation results and those obtained by data mining of motor proteins available in the protein data bank is analyzed. The data mining analysis of motor proteins supports the conformational diversity of the phosphate group of ATP obtained computationally.

Peptide Level Turnover Measurements Enable the Study of Proteoform Dynamics.

Science.gov (United States)

Zecha, Jana; Meng, Chen; Zolg, Daniel Paul; Samaras, Patroklos; Wilhelm, Mathias; Kuster, Bernhard

2018-05-01

The coordination of protein synthesis and degradation regulating protein abundance is a fundamental process in cellular homeostasis. Today, mass spectrometry-based technologies allow determination of endogenous protein turnover on a proteome-wide scale. However, standard dynamic SILAC (Stable Isotope Labeling in Cell Culture) approaches can suffer from missing data across pulse time-points limiting the accuracy of such analysis. This issue is of particular relevance when studying protein stability at the level of proteoforms because often only single peptides distinguish between different protein products of the same gene. To address this shortcoming, we evaluated the merits of combining dynamic SILAC and tandem mass tag (TMT)-labeling of ten pulse time-points in a single experiment. Although the comparison to the standard dynamic SILAC method showed a high concordance of protein turnover rates, the pulsed SILAC-TMT approach yielded more comprehensive data (6000 proteins on average) without missing values. Replicate analysis further established that the same reproducibility of turnover rate determination can be obtained for peptides and proteins facilitating proteoform resolved investigation of protein stability. We provide several examples of differentially turned over splice variants and show that post-translational modifications can affect cellular protein half-lives. For example, N-terminally processed peptides exhibited both faster and slower turnover behavior compared with other peptides of the same protein. In addition, the suspected proteolytic processing of the fusion protein FAU was substantiated by measuring vastly different stabilities of the cleavage products. Furthermore, differential peptide turnover suggested a previously unknown mechanism of activity regulation by post-translational destabilization of cathepsin D as well as the DNA helicase BLM. Finally, our comprehensive data set facilitated a detailed evaluation of the impact of protein

Differentiation of prostate cancer from benign prostate hypertrophy using dual-echo dynamic contrast MR imaging

International Nuclear Information System (INIS)

Muramoto, Satoshi; Uematsu, Hidemasa; Kimura, Hirohiko; Ishimori, Yoshiyuki; Sadato, Norihiro; Oyama, Nobuyuki; Matsuda, Tsuyoshi; Kawamura, Yasutaka; Yonekura, Yoshiharu; Okada, Kenichiro; Itoh, Harumi

2002-01-01

Objective: To investigate the usefulness of dynamic contrast magnetic resonance (MR) imaging in the differentiation of prostate cancer (PC) from benign prostate hypertrophy (BPH). Materials and methods: Eleven PC patients and 13 BPH patients were entered into the analysis. The mean gradient (MG) was calculated from the T2* term-eliminated time-signal intensity curve obtained from dynamic contrast MR data, and the MG of PC and that of BPH were compared. Results: The MG of PC was significantly higher than that of BPH. When the threshold value was set to 1.88% per s for discriminating PC from BPH, the sensitivity, specificity, and accuracy were 100, 85, and 92%, respectively. Conclusion: The MG, which is derived from the T2* term-eliminated time-signal intensity curve, may be a useful index for differentiating PC from BPH

Living biointerfaces based on non-pathogenic bacteria support stem cell differentiation

Science.gov (United States)

Hay, Jake J.; Rodrigo-Navarro, Aleixandre; Hassi, Karoliina; Moulisova, Vladimira; Dalby, Matthew J.; Salmeron-Sanchez, Manuel

2016-02-01

Lactococcus lactis, a non-pathogenic bacteria, has been genetically engineered to express the III7-10 fragment of human fibronectin as a membrane protein. The engineered L. lactis is able to develop biofilms on different surfaces (such as glass and synthetic polymers) and serves as a long-term substrate for mammalian cell culture, specifically human mesenchymal stem cells (hMSC). This system constitutes a living interface between biomaterials and stem cells. The engineered biofilms remain stable and viable for up to 28 days while the expressed fibronectin fragment induces hMSC adhesion. We have optimised conditions to allow long-term mammalian cell culture, and found that the biofilm is functionally equivalent to a fibronectin-coated surface in terms of osteoblastic differentiation using bone morphogenetic protein 2 (BMP-2) added to the medium. This living bacteria interface holds promise as a dynamic substrate for stem cell differentiation that can be further engineered to express other biochemical cues to control hMSC differentiation.

Interplay between H1 and HMGN epigenetically regulates OLIG1&2 expression and oligodendrocyte differentiation.

Science.gov (United States)

Deng, Tao; Postnikov, Yuri; Zhang, Shaofei; Garrett, Lillian; Becker, Lore; Rácz, Ildikó; Hölter, Sabine M; Wurst, Wolfgang; Fuchs, Helmut; Gailus-Durner, Valerie; de Angelis, Martin Hrabe; Bustin, Michael

2017-04-07

An interplay between the nucleosome binding proteins H1 and HMGN is known to affect chromatin dynamics, but the biological significance of this interplay is still not clear. We find that during embryonic stem cell differentiation loss of HMGNs leads to down regulation of genes involved in neural differentiation, and that the transcription factor OLIG2 is a central node in the affected pathway. Loss of HMGNs affects the expression of OLIG2 as well as that of OLIG1, two transcription factors that are crucial for oligodendrocyte lineage specification and nerve myelination. Loss of HMGNs increases the chromatin binding of histone H1, thereby recruiting the histone methyltransferase EZH2 and elevating H3K27me3 levels, thus conferring a repressive epigenetic signature at Olig1&2 sites. Embryonic stem cells lacking HMGNs show reduced ability to differentiate towards the oligodendrocyte lineage, and mice lacking HMGNs show reduced oligodendrocyte count and decreased spinal cord myelination, and display related neurological phenotypes. Thus, the presence of HMGN proteins is required for proper expression of neural differentiation genes during embryonic stem cell differentiation. Specifically, we demonstrate that the dynamic interplay between HMGNs and H1 in chromatin epigenetically regulates the expression of OLIG1&2, thereby affecting oligodendrocyte development and myelination, and mouse behavior. Published by Oxford University Press on behalf of Nucleic Acids Research 2016.

Simulation of quantum dynamics based on the quantum stochastic differential equation.

Science.gov (United States)

Li, Ming

2013-01-01

The quantum stochastic differential equation derived from the Lindblad form quantum master equation is investigated. The general formulation in terms of environment operators representing the quantum state diffusion is given. The numerical simulation algorithm of stochastic process of direct photodetection of a driven two-level system for the predictions of the dynamical behavior is proposed. The effectiveness and superiority of the algorithm are verified by the performance analysis of the accuracy and the computational cost in comparison with the classical Runge-Kutta algorithm.

Molecular Dynamics and Monte Carlo simulations resolve apparent diffusion rate differences for proteins confined in nanochannels

Energy Technology Data Exchange (ETDEWEB)

Tringe, J.W., E-mail: tringe2@llnl.gov [Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA (United States); Ileri, N. [Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA (United States); Department of Chemical Engineering & Materials Science, University of California, Davis, CA (United States); Levie, H.W. [Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA (United States); Stroeve, P.; Ustach, V.; Faller, R. [Department of Chemical Engineering & Materials Science, University of California, Davis, CA (United States); Renaud, P. [Swiss Federal Institute of Technology, Lausanne, (EPFL) (Switzerland)

2015-08-18

Highlights: • WGA proteins in nanochannels modeled by Molecular Dynamics and Monte Carlo. • Protein surface coverage characterized by atomic force microscopy. • Models indicate transport characteristics depend strongly on surface coverage. • Results resolve of a four orders of magnitude difference in diffusion coefficient values. - Abstract: We use Molecular Dynamics and Monte Carlo simulations to examine molecular transport phenomena in nanochannels, explaining four orders of magnitude difference in wheat germ agglutinin (WGA) protein diffusion rates observed by fluorescence correlation spectroscopy (FCS) and by direct imaging of fluorescently-labeled proteins. We first use the ESPResSo Molecular Dynamics code to estimate the surface transport distance for neutral and charged proteins. We then employ a Monte Carlo model to calculate the paths of protein molecules on surfaces and in the bulk liquid transport medium. Our results show that the transport characteristics depend strongly on the degree of molecular surface coverage. Atomic force microscope characterization of surfaces exposed to WGA proteins for 1000 s show large protein aggregates consistent with the predicted coverage. These calculations and experiments provide useful insight into the details of molecular motion in confined geometries.

Differentiable dynamical systems an introduction to structural stability and hyperbolicity

CERN Document Server

Wen, Lan

2016-01-01

This is a graduate text in differentiable dynamical systems. It focuses on structural stability and hyperbolicity, a topic that is central to the field. Starting with the basic concepts of dynamical systems, analyzing the historic systems of the Smale horseshoe, Anosov toral automorphisms, and the solenoid attractor, the book develops the hyperbolic theory first for hyperbolic fixed points and then for general hyperbolic sets. The problems of stable manifolds, structural stability, and shadowing property are investigated, which lead to a highlight of the book, the \\Omega-stability theorem of Smale. While the content is rather standard, a key objective of the book is to present a thorough treatment for some tough material that has remained an obstacle to teaching and learning the subject matter. The treatment is straightforward and hence could be particularly suitable for self-study. Selected solutions are available electronically for instructors only. Please send email to textbooks@ams.org for more informatio...

Duplicate retention in signalling proteins and constraints from network dynamics.

Science.gov (United States)

Soyer, O S; Creevey, C J

2010-11-01

Duplications are a major driving force behind evolution. Most duplicates are believed to fix through genetic drift, but it is not clear whether this process affects all duplications equally or whether there are certain gene families that are expected to show neutral expansions under certain circumstances. Here, we analyse the neutrality of duplications in different functional classes of signalling proteins based on their effects on response dynamics. We find that duplications involving intermediary proteins in a signalling network are neutral more often than those involving receptors. Although the fraction of neutral duplications in all functional classes increase with decreasing population size and selective pressure on dynamics, this effect is most pronounced for receptors, indicating a possible expansion of receptors in species with small population size. In line with such an expectation, we found a statistically significant increase in the number of receptors as a fraction of genome size in eukaryotes compared with prokaryotes. Although not confirmative, these results indicate that neutral processes can be a significant factor in shaping signalling networks and affect proteins from different functional classes differently. © 2010 The Authors. Journal Compilation © 2010 European Society For Evolutionary Biology.

Protein kinase C prevents oligodendrocyte differentiation : Modulation of actin cytoskeleton and cognate polarized membrane traffic

NARCIS (Netherlands)

Baron, W; de Vries, EJ; de Vries, H; Hoekstra, D

1999-01-01

In a previous study, we showed that activation of protein kinase C (PKC) prevents oligodendrocyte differentiation at the pro-oligodendrocyte stage. The present study was undertaken to identify downstream targets of PKC action in oligodendrocyte progenitor cells. Activation of PKC induced the

Value of fast dynamic enhanced MR imaging for the differential diagnosis between ovarian fibroma and subserosal leiomyoma

International Nuclear Information System (INIS)

Shan Jun; Wang Xiaomei; Xu Jianmin; Xu Hongli; Feng Xiaofeng; Gong Jingshan; Ling Rennan; Rao Zibin

2004-01-01

Objective: To study the value of dynamic enhanced MRI in the differential diagnosis between subserosal uterine leiomyoma and ovarian fibroma which were iso-signal and low signal ones on T 2 WI. Methods: Plain and dynamic enhanced MRI findings of 45 patients with histologically proved subserosal leiomyoma or ovarian fibroma were analyzed. There were 35 cases of subserosal leiomyoma and 10 cases of ovarian fibroma. The enhancement index of early phase (100 sec after the contrast medial administration) and the time to peak during 200 sec after the contrast administration (TTP 200 ) were analyzed. Results: On plain scanning, both subserosal leiomyoma and ovarian fibrioma appeared similarly as intrapelvic masses with low signal on T 2 WI. On Gadolinium dynamic enhancement, subserosal leiomyomas showed early and evident enhancement while ovarian fibroma showed delayed and moderate enhancement. The difference of the enhancement index of early phase and TTP 200 was statistically significant (P<0.001). Conclusion: Gadolinium dynamic enhanced MR imaging was helpful in the differential diagnosis between subserosal uterine leiomyoma and ovarian fibroma by the difference in the enhancement pattern

Prediction of methyl-side Chain Dynamics in Proteins

International Nuclear Information System (INIS)

Ming Dengming; Brueschweiler, Rafael

2004-01-01

A simple analytical model is presented for the prediction of methyl-side chain dynamics in comparison with S 2 order parameters obtained by NMR relaxation spectroscopy. The model, which is an extension of the local contact model for backbone order parameter prediction, uses a static 3D protein structure as input. It expresses the methyl-group S 2 order parameters as a function of local contacts of the methyl carbon with respect to the neighboring atoms in combination with the number of consecutive mobile dihedral angles between the methyl group and the protein backbone. For six out of seven proteins the prediction results are good when compared with experimentally determined methyl-group S 2 values with an average correlation coefficient r-bar=0.65±0.14. For the unusually rigid cytochrome c 2 no significant correlation between prediction and experiment is found. The presented model provides independent support for the reliability of current side-chain relaxation methods along with their interpretation by the model-free formalism

Rho GTPases in ameloblast differentiation

Directory of Open Access Journals (Sweden)

Keishi Otsu

2016-05-01

Full Text Available During tooth development, ameloblasts differentiate from inner enamel epithelial cells to enamel-forming cells by modulating the signal pathways mediating epithelial–mesenchymal interaction and a cell-autonomous gene network. The differentiation process of epithelial cells is characterized by marked changes in their morphology and polarity, accompanied by dynamic cytoskeletal reorganization and changes in cell–cell and cell–matrix adhesion over time. Functional ameloblasts are tall, columnar, polarized cells that synthesize and secrete enamel-specific proteins. After deposition of the full thickness of enamel matrix, ameloblasts become smaller and regulate enamel maturation. Recent significant advances in the fields of molecular biology and genetics have improved our understanding of the regulatory mechanism of the ameloblast cell life cycle, mediated by the Rho family of small GTPases. They act as intracellular molecular switch that transduce signals from extracellular stimuli to the actin cytoskeleton and the nucleus. In our review, we summarize studies that provide current evidence for Rho GTPases and their involvement in ameloblast differentiation. In addition to the Rho GTPases themselves, their downstream effectors and upstream regulators have also been implicated in ameloblast differentiation.