Ruler elements in chromatin remodelers set nucleosome array spacing and phasing.

Arrays of regularly spaced nucleosomes dominate chromatin and are often phased by alignment to reference sites like active promoters. How the distances between nucleosomes (spacing), and between phasing sites and nucleosomes are determined remains unclear, and specifically, how ATP-dependent chromatin remodelers impact these features. Here, we used genome-wide reconstitution to probe how Saccharomyces cerevisiae ATP-dependent remodelers generate phased arrays of regularly spaced nucleosomes. We find that remodelers bear a functional element named the 'ruler' that determines spacing and phasing in a remodeler-specific way. We use structure-based mutagenesis to identify and tune the ruler element residing in the Nhp10 and Arp8 modules of the INO80 remodeler complex. Generally, we propose that a remodeler ruler regulates nucleosome sliding direction bias in response to (epi)genetic information. This finally conceptualizes how remodeler-mediated nucleosome dynamics determine stable steady-state nucleosome positioning relative to other nucleosomes, DNA bound factors, DNA ends and DNA sequence elements.

Functional Expression, Purification and Identification of Interaction Partners of PACRG.

PACRG (Parkin co-regulated gene) shares a bi-directional promoter with the Parkinson's disease-associated gene Parkin, but the physiological roles of PACRG have not yet been fully elucidated. Recombinant expression methods are indispensable for protein structural and functional studies. In this study, the coding region of PACRG was cloned to a conventional vector pQE80L, as well as two cold-shock vectors pCold II and pCold-GST, respectively. The constructs were transformed into Escherichia coli (DE3), and the target proteins were overexpressed. The results showed that the cold-shock vectors are more suitable for PACRG expression. The soluble recombinant proteins were purified with Ni2+ chelating column, glutathione S-transferase (GST) affinity chromatography and gel filtration. His6 pull down assay and LC-MS/MS were carried out for identification of PACRG-binding proteins in HEK293T cell lysates, and a total number of 74 proteins were identified as potential interaction partners of PACRG. GO (Gene ontology) enrichment analysis (FunRich) of the 74 proteins revealed multiple molecular functions and biological processes. The highest proportion of the 74 proteins functioned as transcription regulator and transcription factor activity, suggesting that PACRG may play important roles in regulation of gene transcription.

FMNL2 regulates dynamics of fascin in filopodia.

Filopodia are peripheral F-actin-rich structures that enable cell sensing of the microenvironment. Fascin is an F-actin-bundling protein that plays a key role in stabilizing filopodia to support efficient adhesion and migration. Fascin is also highly up-regulated in human cancers, where it increases invasive cell behavior and correlates with poor patient prognosis. Previous studies have shown that fascin phosphorylation can regulate F-actin bundling, and that this modification can contribute to subcellular fascin localization and function. However, the factors that regulate fascin dynamics within filopodia remain poorly understood. In the current study, we used advanced live-cell imaging techniques and a fascin biosensor to demonstrate that fascin phosphorylation, localization, and binding to F-actin are highly dynamic and dependent on local cytoskeletal architecture in cells in both 2D and 3D environments. Fascin dynamics within filopodia are under the control of formins, and in particular FMNL2, that binds directly to dephosphorylated fascin. Our data provide new insight into control of fascin dynamics at the nanoscale and into the mechanisms governing rapid cytoskeletal adaptation to environmental changes. This filopodia-driven exploration stage may represent an essential regulatory step in the transition from static to migrating cancer cells.

Pull-down Assay on Streptavidin Beads and Surface Plasmon Resonance Chips for SWATH-MS-based Interactomics.

BACKGROUND/AIM: Pul-down assay is a popular in vitro method for identification of physical interactors of selected proteins. Here, for the first time, we compared three conventional variants of pull-down assay with the streptavidin-modified surface plasmon resonance (SPR) chips for the detection of PDZ and LIM domain protein 2 (PDLIM2) interaction partners. MATERIALS AND METHODS: PDLIM2 protein-protein interactions were analysed by three variants of pull-down assay on streptavidin beads using LC-MS/MS in "Sequential Window Acquisition of all Theoretical fragment ion spectra (SWATH)" mode and compared with LC-SWATH-MS/MS data from SPR chips. RESULTS: The results showed that (i) the use of SPR chip led to comparable data compared to on-column streptavidin beads, (ii) gravity flow and microflow in wash and elution steps provided better results than centrifugation, and (iii) type and concentration of detergent did not significantly affect the interactome data of cancer-associated PDLIM2. CONCLUSION: Our study supports further application of SPR-based affinity purification with SWATH mass spectrometry for reproducible and controlled characterization of cancer-associated interactomes.

A multi-column plate adapter provides an economical and versatile high-throughput protein purification system.

Protein purification is essential in the study of protein structure and function, and the development of novel therapeutics. Many studies require purifying multiple proteins at once, increasing the demand for improved purification methods. We hypothesized that multiple chromatography columns could be interfaced with a multi-well collection plate for rapid and convenient protein purification without the need of expensive instrumentation. As such, we developed a multi-column plate adapter (MCPA), which provides an economical yet versatile and time efficient, high-throughput protein purification system. The MCPA system simultaneously purified milligrams of different proteins under gravity or under vacuum for faster purification. The MCPA handles up to twenty-four 12 mL columns and multiple MCPA's in sequence allow milligram-scale purification of 96 different samples with relative ease. We also used the MCPA system for large scale affinity purification of four proteins, providing sufficient yields and purity for protein crystallization and biophysical characterization. The MCPA system is ideal for optimizing resin type and volume or any other purification parameter by customizing individual columns during the same purification. The high-throughput and versatile nature of this system should prove to be useful in obtaining adequate amounts of protein for subsequent analyses in any laboratory setting.

Expression and purification of human phosphatase and actin regulator 1 (PHACTR1) in plant-based systems.

Cardiovascular diseases are a prevalent cause of morbidity and mortality especially in industrialized countries. The human phosphatase and actin regulator 1 (PHACTR1) may be involved in such diseases, but its precise regulatory function remains unclear due to the large number of potential interaction partners. The same phenomenon makes this protein difficult to express in mammalian cells, but it is also an intrinsically disordered protein that likely aggregates when expressed in bacteria due to the absence of chaperones. We therefore used a design of experiments approach to test the suitability of three plant-based systems for the expression of satisfactory quantities of recombinant PHACTR1, namely transient expression in tobacco (Nicotiana tabacum) BY-2 plant cell packs (PCPs), whole N. benthamiana leaves and BY-2 cell lysate (BYL). The highest yield was achieved using the BYL: up to 120 mg product kg-1 biomass equivalent within 48 h of translation. This was 1.3-fold higher than transient expression in N. benthamiana together with the silencing inhibitor p19, and 6-fold higher than the PCP system. The presence of Triton X-100 in the extraction buffer increased the recovery of PHACTR1 by 2-200-fold depending on the conditions. PHACTR1 was incompatible with biomass blanching and was stable for less than 16 h in raw plant extracts. Purification using a DDK-tag proved inefficient whereas 15% purity was achieved by immobilized metal affinity chromatography.

Proteomic differences between focal and diffuse traumatic brain injury in human brain tissue.

The early molecular response to severe traumatic brain injury (TBI) was evaluated using biopsies of structurally normal-appearing cortex, obtained at location for intracranial pressure (ICP) monitoring, from 16 severe TBI patients. Mass spectrometry (MS; label free and stable isotope dimethyl labeling) quantitation proteomics showed a strikingly different molecular pattern in TBI in comparison to cortical biopsies from 11 idiopathic normal pressure hydrocephalus patients. Diffuse TBI showed increased expression of peptides related to neurodegeneration (Tau and Fascin, p < 0.05), reduced expression related to antioxidant defense (Glutathione S-transferase Mu 3, Peroxiredoxin-6, Thioredoxin-dependent peroxide reductase; p < 0.05) and increased expression of potential biomarkers (e.g. Neurogranin, Fatty acid-binding protein, heart p < 0.05) compared to focal TBI. Proteomics of human brain biopsies displayed considerable molecular heterogeneity among the different TBI subtypes with consequences for the pathophysiology and development of targeted treatments for TBI.

Depletion of Myofibril-Associated Proteins Using Selective Protein Extraction as a Tool in Cardiac Proteomics.

Muscle tissue poses a particular challenge to proteomic analysis due to a very wide range of protein abundances arising from the dominant expression of myofilament-related proteins. We address this issue by describing proteomic analysis with liquid chromatography-mass spectrometry (LC-MS) and sequential window acquisition of all theoretical mass spectra (SWATH), of guinea pig cardiac tissue prepared in two homogenization buffers: (1) An SDS-based buffer designed to extract "all" tissue proteins and (2) a long-established EDTA-containing buffer thought to preferentially extract non-myofibril-related proteins. We use gene ontology (GO) annotation-based assessment of subcellular localization to indicate if these enriched proteins congregate in the cytoplasm or in organellar lumens. This technique results in the preferential quantitation of less abundant non-myofibrillar proteins and, for future studies, offers the opportunity for more complete analyses of changes in heart tissue protein expression with biological circumstance.

miR-375 Regulates Invasion-Related Proteins Vimentin and L-Plastin.

Invasion is a hallmark of advanced head and neck squamous cell carcinoma (HNSCC). We previously determined that low relative miR-375 expression was associated with poor patient prognosis. HNSCC cells with increased miR-375 expression have lower invasive properties and impaired invadopodium activity. Using stable isotope labeling with amino acids in cell culture and reverse-phase liquid chromatography mass spectrometry, we assessed the impact of miR-375 expression on protein levels in UM-SCC-1 cells. Increased miR-375 expression was associated with down-regulation of proteins involved in cellular assembly and organization, death and survival, and movement. Two invasion-associated proteins, vimentin and L-plastin, were strongly down-regulated by miR-375. Luciferase reporter assays demonstrated that high miR-375 expression reduced vimentin promoter activity, suggesting that vimentin is an indirect target of miR-375. Runt-related transcription factor 1 (RUNX1) is a potential miR-375 direct target, and its knockdown reduced vimentin and L-plastin expression. Data in The Cancer Genome Atlas HNSCC database showed a significant inverse correlation between miR-375 expression and RUNX1, vimentin, and L-plastin RNA expression. These clinical correlations validate our in vitro model findings and support a mechanism in which miR-375 suppresses RUNX1 levels, resulting in reduced vimentin and L-plastin expression. Furthermore, knockdown of RUNX1, L-plastin, and vimentin resulted in significant reductions in cell invasion in vitro, indicating the functional significance of miR-375 regulation of specific proteins involved in HNSCC invasion.

Isolation and characterization of human CapG expressed and post-translationally modified in Pichia pastoris.

CapG is an actin-binding protein, which is overexpressed in a variety of tumors, i.e. breast, ovarian, pancreatic and lung carcinoma. We successfully expressed human CapG in the wild type strain X-33 of the methylotrophic yeast Pichia pastoris (P. pastoris), which does not express endogenous CapG, in order to characterize this protein in more detail. After mechanical cell lysis, debris was centrifuged and the soluble protein was precipitated with ammonium sulfate. This protein pellet was dialyzed and used for CapG purification. Ca2+-dependent exposure of hydrophobic sites allowed single step and selective elution from a Phenyl Sepharose™ matrix. 3.5 mg CapG/10 g wet biomass were isolated and showed a Ca2+-sensitive and dose-dependent capping activity of actin in a fluorometric assay. In P. pastoris, CapG is located at actin patches, actin cables and arranges along the budding neck. The proliferation rate and morphology of the yeast cells are not influenced by the interaction of CapG with actin. The modification pattern of human CapG from P. pastoris and human carcinoma cells is highly similar. We validated most of the known post-translational modifications and found three new phosphorylation and nine new acetylation sites by mass spectrometry. The N-terminus is acetylated or truncated. Truncated CapG is not phosphorylated at the residues S10, T212 and S337. First mutagenesis experiments indicate an N-terminal acetylation dependent C-terminal phosphorylation.

MoVrp1, a putative verprolin protein, is required for asexual development and infection in the rice blast fungus Magnaporthe oryzae.

Endocytosis is a crucial cellular process in eukaryotic cells which involves clathrin and/or adaptor proteins, lipid kinases, phosphatases and the actin cytoskeleton. Verprolin proteins, such as Vrp1 in Saccharomyces cerevisiae, are conserved family proteins that regulate actin binding and endocytosis. Here, we identified and characterized MoVrp1 as the yeast Vrp1 homolog in Magnaporthe oryzae. Deletion of the MoVRP1 gene resulted in defects in vegetative growth, asexual development, and infection of the host plant. The ∆Movrp1 mutants also exhibited decreased extracellular peroxidase and laccase activities and showed defects in colony pigmentation, hyphal surface hydrophobicity, cell wall integrity, autophagy, endocytosis, and secretion of avirulent effector. Our studies provided new evidences that MoVrp1 involved in actin cytoskeleton is important for growth, morphogenesis, cellular trafficking, and fungal pathogenesis.

Isolation of Actin and Actin-Binding Proteins.

Actin-binding proteins mediate and regulate the dynamics of actin and the organization of highly ordered structures of F-actin. Villin is generally expressed in plant cells and is associated with G-actin or F-actin dependent on Ca2+ concentrations. Using a DNase I affinity column chromatography approach, the villin and the G-actin can be isolated from plant material. An outline of this method including the preparation of crude protein extract from plant material, its application on the affinity column, and the successive elution of villin with a solution containing EGTA and then of G-actin with denatured reagents is presented.

A Proteomic-Based Workflow Using Purified Respiratory Syncytial Virus Particles to Identify Cellular Factors as Drug Targets.

The identification of cellular factors that play a role in respiratory syncytial virus (RSV) replication is an alternative strategy in the identification of druggable cellular protein that are essential for RSV replication. In this regard experimental strategies that are able to screen relevant proteins from the vast array of proteins in the cellular milieu will facilitate the identification of potential drug targets. In this chapter we describe a procedure where RSV particles are purified from cells that are permissive for RSV infection, and the protein composition of the purified virus particles characterized using a proteomics-based strategy. This procedure revealed that actin, several actin-binding proteins, and the chaperones HSP70 and HSP90 also co-purified with the virus particles. The relevance of the HSP90 protein to virus replication was then further validated using imaging, gene silencing and by using an established small molecule HSP90 inhibitor.

Development and Application of a High Throughput Protein Unfolding Kinetic Assay.

The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses.

Quantitative Phosphoproteomics Revealed Glucose-Stimulated Responses of Islet Associated with Insulin Secretion.

As central tissue of glucose homeostasis, islet has been an important focus of diabetes research. Phosphorylation plays pivotal roles in islet function, especially in islet glucose-stimulated insulin secretion. A systematic view on how phosphorylation networks were coordinately regulated in this process remains lacking, partially due to the limited amount of islets from an individual for a phosphoproteomic analysis. Here we optimized the in-tip and best-ratio phosphopeptide enrichment strategy and a SILAC-based workflow for processing rat islet samples. With limited islet lysates from each individual rat (20-47 µg), we identified 8539 phosphosites on 2487 proteins. Subsequent quantitative analyses uncovered that short-term (30 min) high glucose stimulation induced coordinate responses of islet phosphoproteome on multiple biological levels, including insulin secretion related pathways, cytoskeleton dynamics, protein processing in ER and Golgi, transcription and translation, and so on. Furthermore, three glucose-responsive phosphosites (Prkar1a pT75pS77 and Tagln2 pS163) from the data set were proved to be correlated with insulin secretion. Overall, we initially gave an in-depth map of islet phosphoproteome regulated by glucose on individual rat level. This was a significant addition to our knowledge about how phosphorylation networks responded in insulin secretion. Also, the list of changed phosphosites was a valuable resource for molecular researchers in diabetes field.

Identification of Tyrosine-Phosphorylated Proteins Upregulated during Epithelial-Mesenchymal Transition Induced with TGF-ß.

The epithelial-to-mesenchymal transition (EMT) is a unique process for the phenotypic changes of tumor cells characterized by a transition from polarized rigid epithelial cells to migrant mesenchymal cells, thus conferring the ability of tumor invasion and metastasis. A major challenge in the treatment of lung adenocarcinoma is to identify early stage patients at a high risk of recurrence or metastasis, thereby permitting the best therapeutic strategy and prognosis. In this study, we used a transforming growth factor-ß (TGF-ß)-induced EMT model to quantitatively identify protein tyrosine phosphorylation during the course of EMT in relation to malignant characteristics of lung adenocarcinoma cells. We performed relative quantitation analysis of tyrosine-phosphorylated peptides in TGF-ß-treated and -untreated lung adenocarcinoma cells and identified tyrosine-phosphorylated proteins that were upregulated in TGF-ß-treated cells. These include tensin-1 (TNS1) phosphorylated on Y1404, hepatocyte growth factor receptor (c-Met) phosphorylated on Y1234, and NT-3 growth factor receptor (TrkC) phosphorylated on Y516. We also found that these protein phosphorylation profiles were specifically observed in tissue samples of patients with poor prognostic lung adenocarcinoma. Tyrosine phosphorylations of these proteins represent possible candidates of prognostic prediction markers for lung adenocarcinoma.

Proteomic profiling of the retinas in a neonatal rat model of oxygen-induced retinopathy with a reproducible ion-current-based MS1 approach.

Investigation of the retina proteome during hypoxia-induced retinal neovascularization is valuable for understanding pathogenesis of retinopathy of prematurity (ROP). Here we employed a reproducible ion-current-based MS1 quantification approach (ICB) to explore the retinal proteomic changes in early stage of ROP in a rat model of oxygen-induced retinopathy (OIR). Retina proteins, which are rich in membrane proteins, were efficiently extracted by a detergent-cocktail and subjected to precipitation/on-pellet-digestion, followed by nano-LC-MS analysis on a 75-cm column with a 7-h gradient. The high reproducibility of sample preparation and chromatography separation enabled excellent peak alignment and contributed to the superior performance of ICB over parallel label-free approaches. In this study, sum-of-intensity with rejection was incorporated to determine the protein ratios. In total, 1325 unique protein groups were quantified from rat retinas (n = 4/group) with at least two distinct peptides at a protein FDR of 1%. Thirty-two significantly altered proteins were observed with confidence, and the elevated glial fibrillary acidic protein and decreased crystalline proteins in OIR retinas agree well with previous studies. Selected key alterations were further validated by Western blot analysis. Interestingly, Rab21/RhoA/ROCK2/moesin signaling pathway was found to be involved in retinal neovascularization of OIR. Moreover, highly elevated annexin A3, a potential angiogenic mediator, was observed in OIR retinas and may serve as a potential therapeutic target. In conclusion, reproducible ICB profiling enabled reliable discovery of many altered mediators and pathways in OIR retinas, thereby providing new insights into molecular mechanisms involved in pathogenesis of ROP.

Recombinant production, purification and crystallization of the Toxoplasma gondii coronin WD40 domain.

Toxoplasma gondii is one of the most widely spread parasitic organisms in the world. Together with other apicomplexan parasites, it utilizes a special actin-myosin motor for its cellular movement, called gliding motility. This actin-based process is regulated by a small set of actin-binding proteins, which in Apicomplexa comprises only 10-15 proteins, compared with >150 in higher eukaryotes. Coronin is a highly conserved regulator of the actin cytoskeleton, but its functions, especially in parasites, have remained enigmatic. Coronins consist of an N-terminal actin-binding ß-propeller WD40 domain, followed by a conserved region, and a C-terminal coiled-coil domain implicated in oligomerization. Here, the WD40 domain and the conserved region of coronin from T. gondii were produced recombinantly and crystallized. A single-wavelength diffraction data set was collected to a resolution of 1.65 Å. The crystal belonged to the orthorhombic space group C2221, with unit-cell parameters a = 55.13, b = 82.51, c = 156.98 Å.

Altered T-cell entry and egress in the absence of Coronin 1A attenuates murine acute graft versus host disease.

Acute graft-versus-host disease (aGvHD) is a major limitation to the use of allogeneic stem cell transplantation for the treatment of patients with relapsed malignant disease. Previous work using animals lacking secondary lymphoid tissue (SLT) suggested that activation of donor T cells in SLT is critically important for the pathogenesis of aGvHD. However, these studies did not determine if impaired migration into, and more importantly, out of SLT, would ameliorate aGvHD. Here, we show that T cells from mice lacking Coronin 1A (Coro 1A(-/-)), an actin-associated protein shown to be important for thymocyte egress, do not mediate acute GvHD. The attenuation of aGvHD was associated with decreased expression of the critical trafficking proteins C-C chemokines receptor type 7 (CCR7) and sphingosine 1 phosphate receptor on donor T cells. This was mediated in part by impaired activation of the canonical NF-κB pathway in the absence of Coro 1A. As a result of these alterations, donor T cells from Coro 1A(-/-) mice were not able to initially traffic to SLT or exit SLT after BM transplantation. However, this alteration did not abrogate the graft-versus-leukemia response. Our data suggest that blocking T-cell migration into and out of SLT is a valid approach to prevent aGvHD.

Impact of actin on adhesion and translocation of Enterococcus faecalis.

This study focuses on the impact of actin on adhesion and translocation of Enterococcus (E.) faecalis OG1RF, E. faecalis Symbioflor(®), and E. faecalis V583. Insight into the role of actin aggregation in the mediation of bacterial adhesion and translocation was provided by a two-chamber translocation assay, which employed Ptk6 cells. Determination of translocation rates, cytochalasin D treatment, and laser scanning confocal microscopic observation revealed actin as a predominant brace for enterococci to pass through the epithelial cell layer. As the three enterococci had moderate adhesion ability to actin, actin-binding proteins were isolated and characterized by LC-MS/MS. The isolated proteins were identified as pyruvate formate lyase, enolase, glyceraldehyde-3-phosphate dehydrogenase, and GroEL. All these proteins belong to two major groups of moonlighting proteins, i.e., proteins, which display additional functions other than their described major biochemical catalysis. Both groups of moonlight proteins were determined to be associated with epithelial cell binding.