Evolution of TNF-Induced Apoptosis Reveals 550 My of Functional Conservation

The Precambrian explosion led to the rapid appearance of most major animal phyla alive today. It has been argued that the complexity of life has steadily increased since that event. Here we challenge this hypothesis through the characterization of apoptosis in reef-building corals, representatives of some of the earliest animals. Bioinformatic analysis reveals that all of the major components of the death receptor pathway are present in coral with high-predicted structural conservation with Homo sapiens. The TNF receptor-ligand superfamilies (TNFRSF/TNFSF) are central mediators of the death receptor pathway, and the predicted proteome of Acropora digitifera contains more putative coral TNFRSF members than any organism described thus far, including humans. This high abundance of TNFRSF members, as well as the predicted structural conservation of other death receptor signaling proteins, led us to wonder what would happen if corals were exposed to a member of the human TNFSF (HuTNFα). HuTNFα was found to bind directly to coral cells, increase caspase activity, cause apoptotic blebbing and cell death, and finally induce coral bleaching. Next, immortalized human T cells (Jurkats) expressing a functional death receptor pathway (WT) and a corresponding Fas-associated death domain protein (FADD) KO cell line were exposed to a coral TNFSF member (AdTNF1) identified and purified here. AdTNF1 treatment resulted in significantly higher cell death (P \u3c 0.0001) in WT Jurkats compared with the corresponding FADD KO, demonstrating that coral AdTNF1 activates the H. sapiens death receptor pathway. Taken together, these data show remarkable conservation of the TNF-induced apoptotic response representing 550 My of functional conservation

An Assay to Monitor HIV-1 Protease Activity for the Identification of Novel Inhibitors in T-Cells

The emergence of resistant HIV strains, together with the severe side-effects of existing drugs and lack of development of effective anti-HIV vaccines highlight the need for novel antivirals, as well as innovative methods to facilitate their discovery. Here, we have developed an assay in T-cells to monitor the proteolytic activity of the HIV-1 protease (PR). The assay is based on the inducible expression of HIV-1 PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the downstream reporter, enhanced green fluorescent protein (eGFP) gene only in the presence of an effective PR Inhibitor (PI). Thus, in this assay, eGFP acts as a biosensor of PR activity, making it ideal for flow cytometry based screening. Furthermore, the assay was developed using retroviral technology in T-cells, thus providing an ideal environment for the screening of potential novel PIs in a cell-type that represents the natural milieu of HIV infection. Clones with the highest sensitivity, and robust, reliable and reproducible reporter activity, were selected. The assay is easily adaptable to other PR variants, a multiplex platform, as well as to high-throughput plate reader based assays and will greatly facilitate the search for novel peptide and chemical compound based PIs in T-cells

Purification of the COP9 Signalosome Complex and Binding Partners from Human T Cells

Abstract The COP9 Signalosome (CSN) is a highly conserved eight subunit protein complex associated with a wide range of essential biological functions in eukaryotic cells, and directly involved in processes including deneddylation, phosphorylation, and ubiquitination. Despite its significant role, very few studies have been undertaken to reveal the interactions between the CSN and its binding partners, and none in human T cells. Here we present a purification method for the CSN and binding proteins via the Streptavidin-Binding Peptide (SBP) fused to CSN Subunit 1 (CSN1). Using this method, coupled with liquid chromatography-mass spectrometry analysis, we identified all eight subunits of the CSN, as well as expected and putative novel binding partners such as a tumor suppressor under the control of Cullin4a-ligase complex; Neurofibromin 2 (Merlin). This work presents a method for fast, reliable, and specific affinity-based purification of a protein complex from a nonadherent cell line. The purification of the CSN and binding partners from T cells can elucidate the roles of CSN in a cell type where it has never been studied before. This proteomic-based approach can broaden our understanding of the functions of the CSN in contexts such as viral-host interactions or immune activation in their natural milieu

Establishment of a clonal T-cell line stably expressing inducible assay elements.

<p>(<b>A</b>) Constructs utilized to generate infectious particles for the transduction of SupT1 cells with the various assay elements (the rtTA, eGFP reporter, and Gal4-based vectors: Gal4, PR/Gal and PRm/Gal4). (<b>B</b>) Flow cytometry analysis of selected clones. Clones expressing the elements of the assay were analyzed with no treatment, with 1µg/mL Dox, or with 1µg/mL Dox and 10µM IDV. (<b>C</b>) Quantification of eGFP expression (left axis and larger bars) and the relative fluorescence intensity (RFI) of each sample (right axis and green bars within larger bars) of clones treated as indicated. The RFI was calculated by normalizing green mean fluorescence intensity (MFI) to the brightest MFI observed (PRm with Dox + IDV).</p

Assay overview.

<p>(<b>A</b>) Wild type Gal4 as control, without Dox. In the absence of Dox, rtTA can not bind to the Tet-Responsive Element (TRE) and consequently, there is no eGFP expression from the reporter construct. In the presence of Dox, rtTA binds to TRE and induces eGFP expression. (<b>B</b>) The PR/Gal4 fusion-based system. In the presence of Dox, PR/Gal4 is expressed; however, its autocatalytic activity results in the separation of the Gal4 domains, resulting in lack of eGFP expression. However, in the presence of PI, the PR/Gal4 fusion remains intact, resulting in the induction of eGFP expression. The same result is expected with an inactive mutant PR, but without the need of inhibitor.</p

Determination of the optimal conditions for the screening of active PIs.

<p>Clonal SupT1 cells harboring inducible Gal4, PRm/Gal4, or PR/Gal4 were activated in a 96 well-plate format under various conditions. (<b>A</b>) Doxycycline titration. Cells were pre-incubated with either DMSO or 10µM IDV and then either left untreated, or activated with 50, 100, 200, 500, 1,000, or 2,000ng/mL of Dox. Cells were analyzed by flow cytometry to determine the number of eGFP positive cells. (<b>B</b>) Time course analysis of eGFP induction in response to Indinavir with or without Dox. Cells were pre-incubated with 10µM IDV and then either left untreated, or activated with 1µg/mL Dox. Cells were analyzed 4, 8, 12, 16, 20, 25, 50 or 75 hours after activation by flow cytometry.</p

Assay response to FDA-approved PIs.

<p>The selected SupT1 clone expressing rtTA, the 5xUAS-eGFP reporter and an inducible PR/Gal4 were incubated with various concentrations of Amprenavir, Atazanavir, Darunavir, Lopinavir, Nelfinavir, Ritonavir, Saquinavir, or Tipranavir, as well as Indinavir, and then activated with 1µg/mL Dox and analyzed 50 hours later by flow cytometry.</p