Dynamic Changes Of Proteins Research Articles

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

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<0.05) were subjected to matrix-assisted laser desorption/ionization time-of-flight/time-of-flight analysis. Fifty-three of these proteins were finally identified using an apple expressed sequence tag database downloaded from Genome Database for Rosaceae and placed into six categories. The categories and the percentage of proteins placed in each category were stress response and defense (49.0%), energy and metabolism (34.0%), fruit ripening and senescence (5.6%), signal transduction (3.8%), cell structure (3.8%) and protein synthesis (3.8%). Proteins involved in several multiple metabolic pathways, including glycolysis, pentose–phosphate pathway, anti-oxidative systems, photosynthesis and cell wall synthesis, were downregulated, especially during the climacteric burst in respiration and during the senescent stages of fruit development. Proteins classified as allergens or involved in cell wall degradation were upregulated during the ripening process. Some protein spots exhibited a mixed pattern (increasing to maximal abundance followed by a decrease), such as 1-aminocyclopropane-1-carboxylate oxidase, L-ascorbate peroxidase and abscisic acid response proteins. The identification of differentially expressed proteins associated with physiological processes identified in the current study provides a baseline of information for understanding the metabolic processes and regulatory mechanisms that occur in climacteric apple fruit during ripening and senescence.

Analyzing the protein assembly and dynamics of the human spliceosome with SILAC.

Quantitative mass spectrometry has become an indispensable tool in proteomic studies. Numerous methods are available and can be applied to approach different issues. In most studies these issues include the quantitative comparison of different cell states, the identification of specific interaction partners or determining degrees of posttranslational modification. In this chapter we describe a SILAC-based quantification in order to analyze dynamic protein changes during the assembly of the human spliceosome on a pre-mRNA in vitro. We provide protocols for assembly of spliceosomes on pre-mRNA (including generation of pre-mRNAs and preparation of nuclear extracts), quantitative mass spectrometry (SILAC labeling, sample preparation), and data analysis to generate timelines for the dynamic protein assembly.

Quantitative Proteomics Reveals the Induction of Mitophagy in Tumor Necrosis Factor-α-activated (TNFα) Macrophages

Macrophages play an important role in innate and adaptive immunity as professional phagocytes capable of internalizing and degrading pathogens to derive antigens for presentation to T cells. They also produce pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) that mediate local and systemic responses and direct the development of adaptive immunity. The present work describes the use of label-free quantitative proteomics to profile the dynamic changes of proteins from resting and TNF-α-activated mouse macrophages. These analyses revealed that TNF-α activation of macrophages led to the down-regulation of mitochondrial proteins and the differential regulation of several proteins involved in vesicle trafficking and immune response. Importantly, we found that the down-regulation of mitochondria proteins occurred through mitophagy and was specific to TNF-α, as other cytokines such as IL-1β and IFN-γ had no effect on mitochondria degradation. Furthermore, using a novel antigen presentation system, we observed that the induction of mitophagy by TNF-α enabled the processing and presentation of mitochondrial antigens at the cell surface by MHC class I molecules. These findings highlight an unsuspected role of TNF-α in mitophagy and expanded our understanding of the mechanisms responsible for MHC presentation of self-antigens.

Identification of Dynamic Changes in Proteins Associated with the Cellular Cytoskeleton after Exposure to Okadaic Acid

Exposure of cells to the diarrhetic shellfish poison, okadaic acid, leads to a dramatic reorganization of cytoskeletal architecture and loss of cell-cell contact. When cells are exposed to high concentrations of okadaic acid (100–500 nM), the morphological rearrangement is followed by apoptotic cell death. Okadaic acid inhibits the broad acting Ser/Thr protein phosphatases 1 and 2A, which results in hyperphosphorylation of a large number of proteins. Some of these hyperphosphorylated proteins are most likely key players in the reorganization of the cell morphology induced by okadaic acid. We wanted to identify these phosphoproteins and searched for them in the cellular lipid rafts, which have been found to contain proteins that regulate cytoskeletal dynamics and cell adhesion. By using stable isotope labeling by amino acids in cell culture cells treated with okadaic acid (400 nM) could be combined with control cells before the isolation of lipid rafts. Protein phosphorylation events and translocations induced by okadaic acid were identified by mass spectrometry. Okadaic acid was shown to regulate the phosphorylation status and location of proteins associated with the actin cytoskeleton, microtubules and cell adhesion structures. A large number of these okadaic acid-regulated proteins have previously also been shown to be similarly regulated prior to cell proliferation and migration. Our results suggest that okadaic acid activates general cell signaling pathways that induce breakdown of the cortical actin cytoskeleton and cell detachment.

Neuroproteomics approach and neurosystems biology analysis: ROCK inhibitors as promising therapeutic targets in neurodegeneration and neurotrauma

Several common degenerative mechanisms and mediators underlying the neuronal injury pathways characterize several neurodegenerative diseases including Alzheimer's, Parkinson's, and Huntington's disease, as well as brain neurotrauma. Such common ground invites the emergence of new approaches and tools to study the altered pathways involved in neural injury alongside with neuritogenesis, an intricate process that commences with neuronal differentiation. Achieving a greater understanding of the impaired pathways of neuritogenesis would significantly help in uncovering detailed mechanisms of axonal regeneration. Among the several agents involved in neuritogenesis are the Rho and Rho kinases (ROCKs), which constitute key integral points in the Rho/ROCK pathway that is known to be disrupted in multiple neuropathologies such as spinal cord injury, traumatic brain injury, and Alzheimer's disease. This in turn renders ROCK inhibition as a promising candidate for therapeutic targets for treatment of neurodegenerative diseases. Among the novel tools to investigate the mechanisms involved in a specific disorder is the use of neuroproteomics/systems biology approach, a growing subfield of bioinformatics aiming to study and establishing a global assessment of the entire neuronal proteome, addressing the dynamic protein changes and interactions. This review aims to examine recent updates regarding how neuroproteomics aids in the understanding of molecular mechanisms of activation and inhibition in the area of neurogenesis and how Rho/ROCK pathway/ROCK inhibitors, primarily Y-27632 and Fasudil compounds, are applied in biological settings, promoting neuronal survival and neuroprotection that has direct future implications in neurotrauma.

Molecular Dynamics Simulations Provide Atomistic Insight into Hydrogen Exchange Mass Spectrometry Experiments

It is now clear that proteins are flexible entities that in solution switch between conformations to achieve their function. Hydrogen/Deuterium Exchange Mass Spectrometry (HX/MS) is an invaluable tool to understand dynamic changes in proteins modulated by cofactor binding, post-transductional modifications, or protein-protein interactions. ERK2MAPK, a protein involved in highly conserved signal transduction pathways of paramount importance for normal cellular function, has been extensively studied by HX/MS. Experiments of the ERK2MAPK in the inactive and active states (in the presence or absence of bound ATP) have provided valuable information on the plasticity of the MAPK domain. However, interpretation of the HX/MS data is difficult, and changes are mostly explained in relation to available X-ray structures, precluding a complete atomic picture of protein dynamics. In the present work, we have used all atom Molecular Dynamics simulations (MD) to provide a theoretical framework for the interpretation of HX/MS data. Our results show that detailed analysis of protein-solvent interaction along the MD simulations allows (i) prediction of the number of protons exchanged for each peptide in the HX/MS experiments, (ii) rationalization of the experimentally observed changes in exchange rates in different protein conditions at the residue level, and (iii) that at least for ERK2MAPK, most of the functionally observed differences in protein dynamics are related to what can be considered the native state conformational ensemble. In summary, the combination of HX/MS experiments with all atom MD simulations emerges as a powerful approach to study protein native state dynamics with atomic resolution.

Enrichment of cell populations in metaphase, anaphase, and telophase by synchronization using nocodazole and blebbistatin: A novel method suitable for examining dynamic changes in proteins during mitotic progression

Mitosis is a continuous process to separate replicated chromosomes into two daughter cells through prophase, metaphase, anaphase, and telophase. Although a number of methods have been established to synchronize cells at different phases of the cell cycle, it is difficult to synchronize cells at the specific phases, anaphase and telophase, during mitosis because of the short duration of anaphase. Here, we show that HeLa S3 cells in anaphase and in telophase are successfully enriched by treatment with a combination of low concentrations of the microtubule-depolymerizing agent nocodazole and the myosin II inhibitor blebbistatin. After 9-h release from thymidine block at G1/S phase, addition of nocodazole at 20ng/ml but not 40ng/ml ensures rapid release from the nocodazole arrest. Subsequently, the cells are cultured in the presence of 50μM blebbistatin for 20 and 50min to enrich cells in anaphase and telophase, respectively. Western blot analysis verifies down-regulation of phospho-histone H3-Ser10, phospho-Aurora A/B/C, and cyclin B1 during M-phase progression. Furthermore, we show how the electrophoretic mobility shifts of the Src-family kinases c-Yes and c-Src can change in each phase of mitosis. These results provide a useful synchronization method for biochemically examining protein dynamics during M-phase progression.

Highly Efficient Fluorescence Quenching with Graphene

Fluorescence quenching is a powerful technique used to obtain information about the dynamic changes of proteins in complex macromolecular systems. In this work, graphene is shown to be a very efficient quencher of fluorescence molecules where the quenching effect was one order of magnitude higher than that of gold. The fluorescence intensity was distance-dependent where increasing the distance between the fluorescence molecule and the graphene surface from 4 to 7 nm increased the fluorescence intensity by a factor of 7.5. This type of distance dependence suggests a nonradiative nature in the energy transfer between the graphene and the fluorophore due to the excitation of an exciton.

Vectorial Network Model (VNM) for Protein Dynamics: An Analytical Tool for Protein Fluctuations

Dynamics play important roles in how proteins function. Coarse grained network model such as Gaussian Network Model (GNM) and Anisotropic Network Model (ANM) are widely used for studying dynamic changes of proteins. It provides an analytical treatment of different eigen modes of protein fluctuations based on the Gaussian assumption. However, GNM and ANM have limitations. For example, detailed mode analysis needed in these models often requires additional ad hoc knowledge for model selection. ANM can only describe mode specific relative movement between residues.

A dynamic model of proteome changes reveals new roles for transcript alteration in yeast

The transcriptome and proteome change dynamically as cells respond to environmental stress; however, prior proteomic studies reported poor correlation between mRNA and protein, rendering their relationships unclear. To address this, we combined high mass accuracy mass spectrometry with isobaric tagging to quantify dynamic changes in ~2500 Saccharomyces cerevisiae proteins, in biological triplicate and with paired mRNA samples, as cells acclimated to high osmolarity. Surprisingly, while transcript induction correlated extremely well with protein increase, transcript reduction produced little to no change in the corresponding proteins. We constructed a mathematical model of dynamic protein changes and propose that the lack of protein reduction is explained by cell-division arrest, while transcript reduction supports redistribution of translational machinery. Furthermore, the transient 'burst' of mRNA induction after stress serves to accelerate change in the corresponding protein levels. We identified several classes of post-transcriptional regulation, but show that most of the variance in protein changes is explained by mRNA. Our results present a picture of the coordinated physiological responses at the levels of mRNA, protein, protein-synthetic capacity, and cellular growth.

Temporal Proteomics Profiling of Lipid Rafts in CCR6-Activated T Cells Reveals the Integration of Actin Cytoskeleton Dynamics

Chemokines orchestrate leukocyte migration toward sites of inflammation and infection and target leukocytes via chemokine receptors such as CCR6, a subfamily of the seven-transmembrane G-protein-coupled receptors. Lipid rafts are cholesterol and sphingolipid-enriched liquid-ordered membrane microdomains thought to serve as scaffolding platforms for signal transduction. To globally understand the dynamic changes of proteins within lipid rafts upon CCR6 activation in T cells, we quantitatively analyzed the time-dependent changes of lipid raft proteome using our recently reported membrane proteomics strategy combining gel-assisted digestion, iTRAQ labeling and LC-MS/MS. To our knowledge, the error-free identification of 852 proteins represents the first data set of the raft proteome in T cells upon chemokine receptor activation, including 354 previously annotated raft proteins and 85 dynamically recruited proteins that are potential raft-associated proteins. The temporal profiles revealed that many proteins involved in the actin cytoskeleton rearrangement are actively recruited into lipid rafts upon CCR6 activation. We further confirmed the proteomics results by Western blotting and used small interfering RNA-mediated knockdown to evaluate their roles upon CCR6 activation. In sum, we employed quantitative proteomic strategy to analyze raft proteome and identified many molecules actively involved in the control of actin assembly and disassembly regulating CCR6 activation-induced cell migration.

Analysis of Cell Surface Proteome Changes via Label-free, Quantitative Mass Spectrometry

We present a mass spectrometry-based strategy for the specific detection and quantification of cell surface proteome changes. The method is based on the label-free quantification of peptide patterns acquired by high mass accuracy mass spectrometry using new software tools and the cell surface capturing technology that selectively enriches glycopeptides exposed to the cell exterior. The method was applied to monitor dynamic protein changes in the cell surface glycoproteome of Drosophila melanogaster cells. The results led to the construction of a cell surface glycoprotein atlas consisting of 202 cell surface glycoproteins of D. melanogaster Kc167 cells and indicated relative quantitative changes of cell surface glycoproteins in four different cellular states. Furthermore we specifically investigated cell surface proteome changes upon prolonged insulin stimulation. The data revealed insulin-dependent cell surface glycoprotein dynamics, including insulin receptor internalization, and linked these changes to intracellular signaling networks.

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L. ‘Moldova’

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape ‘Moldova’ (Vitis vinifera L. ‘Moldova’). Primary calli were initiated on Nitsch and Nitsch (NN) medium supplemented with 1.0 mg·L−1 2,4-D and 0.5 mg·L−1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg·L−1 6-BA and 2 mg·L−1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg·L−1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dynamic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic embryos, including typical epiderma, cotyledon primordium and vascular tissue.

Systems Biology Analysis of Human Primary T Cells Identifies SAP145 as Rate Limiting for the G1→S Phase Transition.

We identified a G0→G1 commitment point in primary human T cells that controls entry into the cell cycle from quiescence. We demonstrated proof of principle that cellular pathways regulating cell cycle progression and effector functions that normally coincide during CD3/CD28 stimulation can be uncoupled experimentally. We have now used systems biology approaches to identify nuclear protein networks in primary human T cells that are regulated during the transition from quiescence into the cell cycle (G0→G1→S-phase). First we sequenced proteins that became bound to chromatin & nuclear matrix in G1 but were not bound in G0 and vice versa by mass spectrometry. Bioinformatic analysis identified 76 proteins specifically bound in G0 not G1 and 254 bound in G1 not G0. 179 of the 254 proteins bound in G1 not G0 (i.e. dynamic protein changes) were mapped to the 55,000 human protein interaction dataset. These are involved in numerous cellular functions, including epigenetics, transcription, RNA splicing & transport, and others. Cell cycle regulated chromatin/matrix binding of a subset was verified by western blotting (2/2 bound in G0 not G1 and 22/23 bound in G1 not G0). One of the proteins induced and bound in G1 was SAP145 (SF3B2). This is a component of the ubiquitous SF3b RNA splicing complex, involved in both major (U2-type) and minor (U12-type) spliceosomes. Since SAP145 is induced during G1 we investigated whether there was a role for SAP145 in regulating cell cycle progression. T cells depleted of SAP145 by siRNA enter G1 from G0 but progress poorly through S phase and die, probably by apoptosis. The same occurs if another component of the SF3B complex, SAP49 (SF3B4) is depleted with siRNA, indicating that the effect is due to depleting the complex rather than the individual SF3B protein. Proteins that are induced during G1 by CD3/CD28 stimulation e.g. cyclin D3, Cdc6 and cdc2 are produced normally when SAP145 is depleted, suggesting that their pre-mRNAs are spliced normally. In contrast, the expression of p107 and cyclin A2 are reduced markedly when SAP145 is depleted. Therefore, a systems biology approach to analysing cell cycle transitions identifies the splicing protein, SAP145 as rate-limiting for the G1 →S phase cell cycle transition but not for the transition from G0→G1.

Steady-state fluorescence quenching applications for studying protein structure and dynamics

Fluorescence quenching methods are useful to obtain information about the conformational and/or dynamic changes of proteins in complex macromolecular systems. In this review steady-state methods are described and the data interpretation is thoroughly discussed. As a special case of fluorescence quenching mechanism, fluorescence resonance energy transfer (FRET) phenomenon is also presented. Application of a FRET based method to characterize the temperature dependence of the flexibility of protein matrix is clearly demonstrated.

Proteomic identification of heat shock protein 70 as a candidate target for enhancing apoptosis induced by farnesyl transferase inhibitor.

Farnesyl transferase inhibitors (FTIs) are novel antitumor drugs with clinical activity. FTIs inhibit cell growth not only by preventing direct Ras farnesylation but also through a Ras-independent pathway. Proteomics has been shown to be a powerful tool to monitor and analyze molecular networks and fluxes within the living cells and to identify the proteins that participate in these networks upon perturbation of the cellular environment. To observe early and dynamic protein changes in the cellular response to FTI in ovarian cancer cells, total proteins were extracted from 2774 cells treated or not with 10 microM manumycin, an FTI, for 3, 6 and 16 h. The proteins in the cells that were differentially expressed following treatment with manumycin for 3, 6 and 16 h were noted by two-dimensional electrophoresis and further identified by peptide mass fingerprinting as stress proteins. Both heat shock protein 70 (HSP70) and altered HSP70 were significantly up-regulated as early as 16 h in 2774 cells after exposure to manumycin. Since HSP70 plays an important role in protecting cells under stress, we treated the 2774 cells with the HSP inhibitor quercetin in combination with FTI. Quercetin dramatically enhanced the manumycin-mediated apoptosis in 2774 cells. Inducible HSP70 by manumycin in surviving ovarian cancer cells was also inhibited by quercetin as demonstrated by enzyme-linked immunosorbent assay. The inhibition of HSP70 by quercetin was correlated with enhancement of manumycin-induced mediated apoptosis in 2774 cells. The inhibition of HSP70 by 50 microM quercetin was also correlated with a decreased expression of procaspase-3 and enhancement of specific cleavage of poly (ADP-ribose) polymerase into apoptotic fragment in 2774 cells treated with manumycin. The interaction between the HSP70 inhibitor and FTI confirms the functional significance of the up-regulation of HSP70 as a protective mechanism against FTI-induced apoptosis and provides the framework for combination treatment of ovarian cancer.