Reduced-representation Phosphosignatures Measured by Quantitative Targeted MS
ABSTRACT: Abelin JG, Patel J, Lu X, Feeney CM, Fagbami L, Creech AL, Hu R, Lam D, Davison D, Pino L, Qiao JW, Kuhn E, Officer A,Li J, Abbatiello S, Subramanian A, Sidman R, Snyder E, Carr SA, Jaffe JD. Mol Cell Proteomics, 2016. Profiling posttranslational modifications represents an alternative dimension to gene expression data in characterizing cellular processes, as genetic processes alone are not sufficient to explain the entirety of biochemical mechanisms or disease etiology. For example, some cellular phenotypes resulting from chemical perturbations are partially or entirely mediated by changes in cell signaling through protein phosphorylation. To access this dimension of cellular information, we sought to develop a common platform on which cellular phosphosignaling responses could be profiled across thousands of samples. To this end, we developed a targeted MS assay that profiles a reduced-representation set of phosphopeptides that we show to be strong indicators of cellular responses to chemical perturbagens.
Project description:Profiling post-translational modifications represents an alternative dimension to gene expression data in characterizing cellular processes. Many cellular responses to drugs are mediated by changes in cellular phosphosignaling. We sought to develop a common platform on which phosphosignaling responses could be profiled across thousands of samples, and created a targeted MS assay that profiles a reduced-representation set of phosphopeptides that we show to be strong indicators of responses to chemical perturbagens.To develop the assay, we investigated the coordinate regulation of phosphosites in samples derived from three cell lines treated with 26 different bioactive small molecules. Phosphopeptide analytes were selected from these discovery studies by clustering and picking 1 to 2 proxy members from each cluster. A quantitative, targeted parallel reaction monitoring assay was developed to directly measure 96 reduced-representation probes. Sample processing for proteolytic digestion, protein quantification, peptide desalting, and phosphopeptide enrichment have been fully automated, making possible the simultaneous processing of 96 samples in only 3 days, with a plate phosphopeptide enrichment variance of 12%. This highly reproducible process allowed ?95% of the reduced-representation phosphopeptide probes to be detected in ?200 samples.The performance of the assay was evaluated by measuring the probes in new samples generated under treatment conditions from discovery experiments, recapitulating the observations of deeper experiments using a fraction of the analytical effort. We measured these probes in new experiments varying the treatments, cell types, and timepoints to demonstrate generalizability. We demonstrated that the assay is sensitive to disruptions in common signaling pathways (e.g. MAPK, PI3K/mTOR, and CDK). The high-throughput, reduced-representation phosphoproteomics assay provides a platform for the comparison of perturbations across a range of biological conditions, suitable for profiling thousands of samples. We believe the assay will prove highly useful for classification of known and novel drug and genetic mechanisms through comparison of phosphoproteomic signatures.
Project description:Previous works showed that immunization with saliva from Lutzomyia intermedia, a vector of Leishmania braziliensis, does not protect against experimental infection. However, L. braziliensis is also transmitted by Lutzomyia whitmani, a sand fly species closely related to Lu. intermedia. Herein we describe the immune response following immunization with Lu. whitmani saliva and the outcome of this response after L. braziliensis infection.BALB/c mice immunized with Lu. whitmani saliva developed robust humoral and cellular immune responses, the latter characterized by an intense cellular infiltrate and production of IFN-? and IL-10, by both CD4+ and CD8+ cells. Mice immunized as above and challenged with L. braziliensis plus Lu. whitmani saliva displayed significantly smaller lesions and parasite load at the challenge site. This protection was associated with a higher (p<0.05) IFN-? production in response to SLA stimulation. Long-term persisting immunity was also detected in mice immunized with Lu. whitmani saliva. Furthermore, individuals residing in an endemic area for cutaneous leishmaniasis (CL) presented antibody responses to Lu. whitmani saliva. However CL patients, with active lesions, displayed a lower humoral response to Lu. whitmani saliva compared to individuals with subclinical Leishmania infection.Pre-exposure to Lu. whitmani saliva induces protection against L. braziliensis in a murine model. We also show that Lu. whitmani salivary proteins are immunogenic in naturally exposed individuals. Our results reinforce the importance of investigating the immunomodulatory effect of saliva from different species of closely related sand flies.
Project description:BACKGROUND:Sheath blight (SB), caused by Rhizoctonia solani, is a common rice disease worldwide. Currently, rice cultivars with robust resistance to R. solani are still lacking. To provide theoretic basis for molecular breeding of R. solani-resistant rice cultivars, the changes of transcriptome profiles in response to R. solani infection were compared between a moderate resistant cultivar (Yanhui-888, YH) and a susceptible cultivar (Jingang-30, JG). RESULTS:In the present study, 3085 differentially express genes (DEGs) were detected between the infected leaves and the control in JG, with 2853 DEGs in YH. A total of 4091 unigenes were significantly upregulated in YH than in JG before infection, while 3192 were significantly upregulated after infection. Further analysis revealed that YH and JG showed similar molecular responses to R. solani infection, but the responses were earlier in JG than in YH. Expression levels of trans-cinnamate 4-monooxygenase (C4H), ethylene-insensitive protein 2 (EIN2), transcriptome factor WRKY33 and the KEGG pathway plant-pathogen interaction were significantly affected by R. solani infection. More importantly, these components were all over-represented in YH cultivar than in JG cultivar before and/or after infection. CONCLUSIONS:These genes possibly contribute to the higher resistance of YH to R. solani than JG and were potential target genes to molecularly breed R. solani-resistant rice cultivar.
Project description:Although transcriptional induction of stress genes constitutes a major cellular defense program against a variety of stressors, posttranslational control directly regulating the activities of preexisting stress proteins provides a faster-acting alternative response. We propose that posttranslational control is a general adaptive mechanism operating in many stress pathways. Here with the aid of computational models, we first show that posttranslational control fulfills two roles: (1) handling small, transient stresses quickly and (2) stabilizing the negative feedback transcriptional network. We then review the posttranslational control pathways for major stress responses-oxidative stress, metal stress, hyperosmotic stress, DNA damage, heat shock, and hypoxia. Posttranslational regulation of stress protein activities occurs by reversible covalent modifications, allosteric or non-allosteric enzymatic regulations, and physically induced protein structural changes. Acting in feedback or feedforward networks, posttranslational control may establish a threshold level of cellular stress. Sub-threshold stresses are handled adequately by posttranslational control without invoking gene transcription. With supra-threshold stress levels, cellular homeostasis cannot be maintained and transcriptional induction of stress genes and other gene programs, eg, those regulating cell metabolism, proliferation, and apoptosis, takes place. The loss of homeostasis with consequent changes in cellular function may lead to adverse cellular outcomes. Overall, posttranslational and transcriptional control pathways constitute a stratified cellular defense system, handling stresses coherently across time and intensity. As cell-based assays become a focus for chemical testing anchored on toxicity pathways, examination of proteomic and metabolomic changes as a result of posttranslational control occurring in the absence of transcriptomic alterations deserves more attention.
Project description:Understanding the glass transition requires getting the picture of the dynamical processes that intervene in it. Glass-forming liquids show a characteristic decoupling of relaxation processes when they are cooled down towards the glassy state. The faster (?JG) process is still under scrutiny, and its full explanation necessitates information at the microscopic scale. To this aim, nuclear ?-resonance time-domain interferometry (TDI) has been utilized to investigate 5-methyl-2-hexanol, a hydrogen-bonded liquid with a pronounced ?JG process as measured by dielectric spectroscopy. TDI probes in fact the center-of-mass, molecular dynamics at scattering-vectors corresponding to both inter- and intra-molecular distances. Our measurements demonstrate that, in the undercooled liquid phase, the ?JG relaxation can be visualized as a spatially-restricted rearrangement of molecules within the cage of their closest neighbours accompanied by larger excursions which reach out at least the inter-molecular scale and are related to cage-breaking events. In-cage rattling and cage-breaking processes therefore coexist in the ?JG relaxation.
Project description:Cancer cells rely on the chaperone heat shock protein 70 (Hsp70) for survival and proliferation. Recently, benzothiazole rhodacyanines have been shown to bind an allosteric site on Hsp70, interrupting its binding to nucleotide-exchange factors (NEFs) and promoting cell death in breast cancer cell lines. However, proof-of-concept molecules, such as JG-98, have relatively modest potency (EC50 ? 0.7-0.4 ?M) and are rapidly metabolized in animals. Here, we explored this chemical series through structure- and property-based design of ?300 analogs, showing that the most potent had >10-fold improved EC50 values (?0.05 to 0.03 ?M) against two breast cancer cells. Biomarkers and whole genome CRISPRi screens confirmed members of the Hsp70 family as cellular targets. On the basis of these results, JG-231 was found to reduce tumor burden in an MDA-MB-231 xenograft model (4 mg/kg, ip). Together, these studies support the hypothesis that Hsp70 may be a promising target for anticancer therapeutics.
Project description:The responses of cells to their surroundings are mediated by the binding of cell surface proteins (CSPs) to extracellular signals. Such processes are regulated via dynamic changes in the structure, composition, and expression levels of CSPs. In this study, we demonstrate the possibility of decorating bacteria with artificial, self-assembled receptors that imitate the dynamic features of CSPs. We show that the local concentration of these receptors on the bacterial membrane and their structure can be reversibly controlled using suitable chemical signals, in a way that resembles changes that occur with CSP expression levels or posttranslational modifications (PTMs), respectively. We also show that these modifications can endow the bacteria with programmable properties, akin to the way CSP responses can induce cellular functions. By programming the bacteria to glow, adhere to surfaces, or interact with proteins or mammalian cells, we demonstrate the potential to tailor such biomimetic systems for specific applications.
Project description:Chemical modifications of histone proteins directly and indirectly affect chromatin structure and thereby contribute to the multilayered control of diverse DNA-based processes. A recent study in Nature enriches this list of enzyme-dependent posttranslational histone marks by H2A glutamine methylation that appears to be dedicated to only one specific cellular process, the regulation of nucleolar rDNA transcription.
Project description:The representation of land use (LU) in meteorological modeling strongly influences the simulation of fluxes of heat, moisture, and momentum; affecting the accuracy of 2-m temperature and precipitation. Here, the Weather Research and Forecasting (WRF) model is used with the Noah land surface model to compare a mosaic approach, which accounts for subgrid scale variability of LU types, to the default option which only considers the dominant category in each grid cell. Three-year historical dynamically downscaled WRF simulations are generated using a 12-km domain over the contiguous U.S. to assess the sensitivities to using mosaic LU and to changes to parameters associated with LU and soil categories. Compared to dominant LU, mosaic LU features decreased coverage of forest and agricultural types and increased low-density urban LU throughout much of the eastern and central U.S. However, highly urbanized areas show the opposite trend, as mosaic LU represents partial greenspace within areas that are exclusively urban within dominant LU. Mosaic LU results in widespread increases in sensible heat fluxes and 2-m temperatures, with reductions in latent heat flux, 2-m mixing ratio, and monthly precipitation across the central and eastern U.S. These changes exacerbate an existing warm bias found with dominant LU but reduce overestimations of precipitation. Highly urbanized areas in the eastern U.S. tend to have cooler, more realistic temperatures with mosaic LU relative to dominant LU. A pair of runs with updated surface parameters corroborates these results. Overall, differences between the simulations are largely attributable to their representations of urban LU.
Project description:The revolutions in biotechnology and information technology have produced clinical data, which complement biological data. These data enable detailed descriptions of various healthy and diseased states and responses to therapies. For the investigation of the physiology and pathology of the immune responses, computer and mathematical models have been used in the last decades, enabling the representation of biological processes. In this modeling effort, a major issue is represented by the communication between models that work at cellular and molecular level, that is, multiscale representation. Here we sketch some attempts to model immune system dynamics at both levels.