1,721,109 research outputs found
Molecular Analysis of TTP, a Key Regulator of TNFα Activity
No full textThe immediate-early protein tristetraprolin (TTP) is induced by numerous extracellular stimuli and during some apoptotic events. TTP and the related proteins TIS11b and TIS11d (TTP/TIS11 proteins) are members of a family with highly conserved Cys3His zinc fingers that are associated with RNA binding. In mice, lack of TTP causes an inflammatory syndrome that is mediated by TNFα 1, and evidence indicates that TTP can bind and destabilize TNFα and other cytokine mRNAs2. In vitro, all three TTP/TIS11 proteins possess this activity and it is present within the zinc finger region3. Several precedents suggest however that TTP/TIS11 proteins may also be implicated in survival, growth, or apoptotic pathways; a model we have addressed in this study.Breast Cancer Research Program (U.S.) (17-94-J-4063)National Institutes of Health (U.S.) (Grant PO1 HD17461)Arthritis Foundation (Postdoctoral Fellowship
Salvaging the septic heart through targeting the IL-6/p38 MAPK signaling network
Get PDFDepression of myocardial function during severe sepsis, which currently accounts for approx. 200,000 deaths/year in the United States (1), is characterized by a decrease in contractility and a poor response to fluid therapy (2). Since the md-1980s it has been recognized that the decreased cardiac function, which undoubtedly contributes to the overall pathophysiology of the septic state, does not arise from factors that are intrinsic to the myocardium, but instead results from the presence of circulating myocardial depressant factors (3, 4). Since much of the massive inflammation and multi-organ dysfunction in sepsis result from the secretion of various cytokines, it was long suspected that these proteins were also responsible, at least in part, for the observed myocardial dysfunction, although their identification, and the molecular basis for their effects on myocyte function were poorly understood
Fixin' to Divide
No full textIn this issue of Molecular Cell, Yata et al. (2012) show that the mitotic kinase and cell-cycle regulator Plk1 can directly stimulate the DNA repair process, providing a potential mechanism of crosstalk between DNA repair and cell-cycle signaling
Signaling netwErks get the global treatment
No full textTwo landmark studies of cell signaling, by RNA interference and phosphoproteomics, provide complementary global views of the pathways downstream of receptor kinases, including those regulated by Erks
Selective Mitogen Activated Protein Kinase Activity Sensors through the Application of Directionally Programmable D Domain Motifs
No full textAccurate and quantitative methods for measuring the dynamic fluctuations of protein kinase activities are critically needed as diagnostic tools and for the evaluation of kinase-targeted inhibitors, which represent a major therapeutic development area in the treatment of cancer and other diseases. In particular, rapid and economical methods that utilize simple instrumentation and provide quantitative data in a high throughput format will have the most impact on basic research in systems biology and medicine. There are over 500 protein kinases in the human kinome. Among these, the mitogen activated protein (MAP) kinases are recognized to be central players in key cellular signaling events and are associated with essential processes including growth, proliferation, differentiation, migration, and apoptosis. The major challenge with MAP kinase sensor development is achieving high selectivity since these kinases rely acutely on secondary interactions distal to the phosphorylation site to impart substrate specificity. Herein we describe the development and application of selective sensors for three MAP kinase subfamilies, ERK1/2, p38α/β, and JNK1/2/3. The new sensors are based on a modular design, which includes a sensing element that exploits a sulfonamido-oxine (Sox) fluorophore for reporting phosphorylation, a recognition and specificity element based on reported docking domain motifs and a variable linker, which can be engineered to optimize the intermodule distance and relative orientation. Following rigorous validation, the capabilities of the new sensors are exemplified through the quantitative analysis of the target MAP kinases in breast cancer progression in a cell culture model, which reveals a strong correlation between p38α/β activity and increased tumorgenicity.National Institutes of Health (U.S.) (NIH Ruth L. Kirschstein National Research Service Award (F32 GM102992))David H. Koch Institute for Integrative Cancer Research at MIT (Koch Institute Bridge Project Grant)National Cancer Institute (U.S.) (Integrated Cancer Biology Program at MIT (NIH U54-CA112967))National Institutes of Health (U.S.) (NIH GM104047)National Institutes of Health (U.S.) (NIH ES015339
Robust co-regulation of tyrosine phosphorylation sites on proteins reveals novel protein interactions
No full textCell signaling networks propagate information from extracellular cues via dynamic modulation of protein–protein interactions in a context-dependent manner. Networks based on receptor tyrosine kinases (RTKs), for example, phosphorylate intracellular proteins in response to extracellular ligands, resulting in dynamic protein–protein interactions that drive phenotypic changes. Most commonly used methods for discovering these protein–protein interactions, however, are optimized for detecting stable, longer-lived complexes, rather than the type of transient interactions that are essential components of dynamic signaling networks such as those mediated by RTKs. Substrate phosphorylation downstream of RTK activation modifies substrate activity and induces phospho-specific binding interactions, resulting in the formation of large transient macromolecular signaling complexes. Since protein complex formation should follow the trajectory of events that drive it, we reasoned that mining phosphoproteomic datasets for highly similar dynamic behavior of measured phosphorylation sites on different proteins could be used to predict novel, transient protein–protein interactions that had not been previously identified. We applied this method to explore signaling events downstream of EGFR stimulation. Our computational analysis of robustly co-regulated phosphorylation sites, based on multiple clustering analysis of quantitative time-resolved mass-spectrometry phosphoproteomic data, not only identified known sitewise-specific recruitment of proteins to EGFR, but also predicted novel, a priori interactions. A particularly intriguing prediction of EGFR interaction with the cytoskeleton-associated protein PDLIM1 was verified within cells using co-immunoprecipitation and in situ proximity ligation assays. Our approach thus offers a new way to discover protein–protein interactions in a dynamic context- and phosphorylation site-specific manner.National Institutes of Health (U.S.) (Grant U54-CA112967)National Institutes of Health (U.S.) (Grant R01-CA96504)National Institutes of Health (U.S.) (Grant P50-GM68762)National Institutes of Health (U.S.) (Grant R01 ES-015339
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Human neutrophil elastase mediates fibrinolysis shutdown through competitive degradation of plasminogen and generation of angiostatin
No full textBACKGROUND A subset of trauma patients undergo fibrinolysis shutdown rather than pathologic hyperfibrinolysis, contributing to organ failure. The molecular basis for fibrinolysis shutdown in trauma is incompletely understood. Elastase released from primed/activated human neutrophils (HNE) has historically been described as fibrin(ogen)olytic. However, HNE can also degrade plasminogen (PLG) to angiostatin (ANG), retaining the kringle domains but not the proteolytic function, and could thereby compete for generation of active plasmin by tissue plasminogen activator (tPA). We hypothesized that HNE can drive fibrinolysis shutdown rather than fibrinolysis. METHODS Turbidometry was performed using light scatter (λ = 620 nm) in a purified fibrinogen + PLG system and in healthy citrate plasma clotted with Ca2+/thrombin ± tPA, ±HNE, and ±ANG to evaluate HNE effects on fibrinolysis, quantified by time to transition midpoint (Tm). ΔTmfrom control is reported as percent of control ±95% CI. Purified HNE coincubated with PLG or tPA was analyzed by western blot to identify cleavage products. Exogenous HNE was mixed ex vivo with healthy volunteer blood (n = 7) and used in TEG ± tPA to evaluate effects on fibrinolysis. RESULTS HNE did not cause measurable fibrinolysis on fibrin clots, clotted plasma, or whole blood as assessed by turbidometry or TEG in the absence of tPA. Upon tPA treatment, all three methods of evaluating fibrinolysis showed delays and decreases in fibrinolysis caused by HNE relative to control: fibrin clot turbidometry ΔTm= 110.7% (CI 105.0-116.5%), clotted citrate plasma (n = 6 healthy volunteers) ΔTm= 126.1% (CI 110.4-141.8%), and whole blood native TEG (n = 7 healthy volunteers) with ΔLY30 = 28% (p = 0.043). Western blot analysis of HNE-PLG co-incubation confirmed that HNE generates angiostatin K1-3, and plasma turbidity assays treated with angiostatin K1-3 delayed fibrinolysis. CONCLUSION HNE degrades PLG and generates angiostatin K1-3, which predominates over HNE cleavage of fibrin(ogen). These findings suggest that neutrophil release of elastase may underlie trauma-induced fibrinolytic shutdown.National Institutes of Health (U.S.) (Grant F32-HL134244)National Institutes of Health (U.S.) (Grant L30-GM120751United States. Department of Defense (Grant 151953
NetworKIN: a resource for exploring cellular phosphorylation networks
Get PDFProtein kinases control cellular responses by phosphorylating specific substrates. Recent proteome-wide mapping of protein phosphorylation sites by mass spectrometry has discovered thousands of in vivo sites. Systematically assigning all 518 human kinases to all these sites is a challenging problem. The NetworKIN database (http://networkin.info) integrates consensus substrate motifs with context modelling for improved prediction of cellular kinase–substrate relations. Based on the latest human phosphoproteome from the Phospho.ELM and PhosphoSite databases, the resource offers insight into phosphorylation-modulated interaction networks. Here, we describe how NetworKIN can be used for both global and targeted molecular studies. Via the web interface users can query the database of precomputed kinase–substrate relations or obtain predictions on novel phosphoproteins. The database currently contains a predicted phosphorylation network with 20 224 site-specific interactions involving 3978 phosphoproteins and 73 human kinases from 20 families.Genome Canada (through Ontario Genomics Institute)National Institutes of Health (U.S.) (U54-CA112967)National Institutes of Health (U.S.) (GM60594)European Community’s Human Potential Programme (BioSapiens Network of Excellence (contract number LSHG-CT-2003-503265))European Community’s Human Potential Programme (ADIT Integrated Project (contract number LSHB-CT-2005511065)
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