Chemoproteomics reveals Toll-like receptor fatty acylation

Partial funding for Open Access provided by The Ohio State University Open Access Fund.Background: Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells. Results: A total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2 S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased inflammatory response to microbial ligands. Conclusions: This work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. Spalmitoylation of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue for modulation of TLR2 inflammatory activity.This work was supported by funding from the NIH/NIAID (grant R00AI095348 to J.S.Y.), the NIH/NIGMS (R01GM087544 to HCH), and the Ohio State University Public Health Preparedness for Infectious Diseases (PHPID) program. NMC is supported by the Ohio State University Systems and Integrative Biology Training Program (NIH/NIGMS grant T32GM068412). BWZ is a fellow of the National Science Foundation Graduate Research Fellowship Program (DGE-0937362)

Discovery of 1,3-Diaminobenzenes as Selective Inhibitors of Platelet Activation at the PAR1 Receptor

A high-throughput screen of the NIH-MLSMR compound collection, along with a series of secondary assays to identify potential targets of hit compounds, previously identified a 1,3-diaminobenzene scaffold that targets protease-activated receptor 1 (PAR1). We now report additional structure–activity relationship (SAR) studies that delineate the requirements for activity at PAR1 and identify plasma-stable analogues with nanomolar inhibition of PAR1-mediated platelet activation. Compound 4 was declared as a probe (ML161) with the NIH Molecular Libraries Program. This compound inhibited platelet aggregation induced by a PAR1 peptide agonist or by thrombin but not by several other platelet agonists. Initial studies suggest that ML161 is an allosteric inhibitor of PAR1. These findings may be important for the discovery of antithrombotics with an improved safety profile

Identification of a Novel Binding Partner of Phospholipase Cβ1: Translin-Associated Factor X

Mammalian phospholipase Cβ1 (PLCβ1) is activated by the ubiquitous Gαq family of G proteins on the surface of the inner leaflet of plasma membrane where it catalyzes the hydrolysis of phosphatidylinositol 4,5 bisphosphate. In general, PLCβ1 is mainly localized on the cytosolic plasma membrane surface, although a substantial fraction is also found in the cytosol and, under some conditions, in the nucleus. The factors that localize PLCβ1in these other compartments are unknown. Here, we identified a novel binding partner, translin-associated factor X (TRAX). TRAX is a cytosolic protein that can transit into the nucleus. In purified form, PLCβ1 binds strongly to TRAX with an affinity that is only ten-fold weaker than its affinity for its functional partner, Gαq. In solution, TRAX has little effect on the membrane association or the catalytic activity of PLCβ1. However, TRAX directly competes with Gαq for PLCβ1 binding, and excess TRAX reverses Gαq activation of PLCβ1. In C6 glia cells, endogenous PLCβ1 and TRAX colocalize in the cytosol and the nucleus, but not on the plasma membrane where TRAX is absent. In Neuro2A cells expressing enhanced yellow and cyano fluorescent proteins (i.e., eYFP- PLCβ1 and eCFP-TRAX), Förster resonance energy transfer (FRET) is observed mostly in the cytosol and a small amount is seen in the nucleus. FRET does not occur at the plasma membrane where TRAX is not found. Our studies show that TRAX, localized in the cytosol and nucleus, competes with plasma-membrane bound Gαq for PLCβ1 binding thus stabilizing PLCβ1 in other cellular compartments

Proteomic Analysis of S-Acylated Proteins in Human B Cells Reveals Palmitoylation of the Immune Regulators CD20 and CD23

S-palmitoylation is a reversible post-translational modification important for controlling the membrane targeting and function of numerous membrane proteins with diverse roles in signalling, scaffolding, and trafficking. We sought to identify novel palmitoylated proteins in B lymphocytes using acyl-biotin exchange chemistry, coupled with differential analysis by liquid-chromatography tandem mass spectrometry. In total, we identified 57 novel palmitoylated protein candidates from human EBV-transformed lymphoid cells. Two of them, namely CD20 and CD23 (low affinity immunoglobulin epsilon Fc receptor), are immune regulators that are effective/potential therapeutic targets for haematological malignancies, autoimmune diseases and allergic disorders. Palmitoylation of CD20 and CD23 was confirmed by heterologous expression of alanine mutants coupled with bioorthogonal metabolic labeling. This study demonstrates a new subset of palmitoylated proteins in B cells, illustrating the ubiquitous role of protein palmitoylation in immune regulation

Sustaining Clinical Programs During Difficult Economic Times: A Case Series from the Hospital Elder Life Program

OBJECTIVES: To explore strategies used by clinical programs to justify operations to decision-makers using the example of the Hospital Elder Life Program (HELP), an evidence-based, cost-effective program to improve care for hospitalized older adults. DESIGN: Qualitative study design using 62 in-depth, semistructured interviews conducted with HELP staff members and hospital administrators between September 2008 and August 2009. SETTING: Nineteen HELP sites in hospitals across the United States and Canada that had been recruiting patients for at least 6 months. PARTICIPANTS: HELP staff and hospital administrators. MEASUREMENTS: Participant experiences sustaining the program in the face of actual or perceived financial threats, with a focus on factors they believe are effective in justifying the program to decision-makers in the hospital or health system. RESULTS: Using the constant comparative method, a standard qualitative analysis technique, three major themes were identified across interviews. Each focuses on a strategy for successfully justifying the program and securing funds for continued operations: interact meaningfully with decision-makers, including formal presentations that showcase operational successes and informal means that highlight the benefits of HELP to the hospital or health system; document day-to-day, operational successes in metrics that resonate with decision-maker priorities; and garner support from influential hospital staff that feed into administrative decision-making, particularly nurses and physicians. CONCLUSION: As clinical programs face financially challenging times, it is important to find effective ways to justify their operations to decision-makers. Strategies described here may help clinically effective and cost-effective programs sustain themselves and thus may help improve care in their institutions

NICE to HELP: Operationalizing National Institute for Health and Clinical Excellence Guidelines to Improve Clinical Practice

The National Institute for Health and Clinical Excellence (NICE) in the United Kingdom developed guidelines for the diagnosis, prevention, and management of delirium in July 2010 that included 10 recommendations for delirium prevention. The Hospital Elder Life Program (HELP) is a targeted multicomponent strategy that has proven effective and cost-effective at preventing functional and cognitive decline in hospitalized older persons. HELP provided much of the basis for seven of the NICE recommendations. Given interest by new HELP sites to meet NICE guidelines, three new protocols addressing hypoxia, infection, and pain that were not previously included in the HELP program were developed. In addition, the NICE dehydration guideline included constipation, which was not specifically addressed in the HELP protocols. This project describes the systematic development of three new protocols (hypoxia, infection, pain) and the expansion of an existing HELP protocol (constipation and dehydration) to achieve alignment between the HELP protocols and NICE guidelines. Following the Institute of Medicine recommendations for developing trustworthy guidelines, an interdisciplinary group of experts conducted a systematic review of current literature, rated the quality of the evidence, developed intervention protocols based on the highest-quality evidence, and submitted the protocols first to internal review and then to external review by an interdisciplinary panel of experts. The protocols were revised based on the review process and incorporated into the HELP materials. Inclusion of these protocols enhances the scope of the HELP program and allows fulfillment of NICE guideline recommendations for delirium prevention. The rigorous process applied may provide a useful example for updating existing guidelines or protocols that may be applicable to a broad range of clinical applications