Browsing by Subject "Transcription Factor AP-1"
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Item ADAP1 Promotes Latent HIV-1 Reactivation by Tuning the KRAS-ERK-AP-1 Signaling-Transcriptional Axis(December 2021) Ramirez, Nora-Guadalupe Piña; Schoggins, John W.; D'Orso, Iván; Pfeiffer, Julie K.; Alto, NealImmune stimulation fuels cell signaling-transcriptional programs that induce biological responses to eliminate virus-infected cells. Yet, retroviruses that integrate into host cell chromatin, such as HIV-1, co-opt these programs to switch between latent and reactivated states. However, many regulatory mechanisms are still unfolding. As such, here I take advantage of the unique intrinsic reliance HIV-1 has on host cell signaling-transcriptional programs to discover undescribed cell signaling regulators. Specifically, I implemented a functional screening platform, given HIV-1 gene expression relies on CD4+ T cell activation state, to identify host factors modulating CD4+ T cell signaling-transcriptional axes and consequently HIV-1 fate. Among the hits, I focus on ADAP1 (ArfGAP with Dual PH Domains 1), a previously thought neuro-restricted factor, and discover it is an amplifier of select human CD4+ T cell signaling programs. Using physiological models, I characterize ADAP1 expression is low in naïve and memory CD4+ T cells, but largely induced upon immune stimulation where it interacts with the immune signalosome. Using complementary biochemical and cellular assays, I demonstrate ADAP1 directly stimulates the GTPase activity of KRAS to amplify CD4+ T cell signaling through targeted activation of ERK-AP-1 axis. In primary CD4+ T cells which I have genetically ablated ADAP1, I show loss of ADAP1 function blunts gene expression programs in response to stimulation thereby reducing CD4+ T cell expansion and dampening latent HIV-1 reactivation. Supporting the impact of these findings, I propose the reduced CD4+ T cell programs and proliferation upon ADAP1 loss validates Genome-wide Association Studies linking ADAP1 single nucleotide polymorphisms in non-coding enhancers to an altered T lymphocyte count trait, potentially attributed to ADAP1 haploinsufficiency. Through these combined experimental approaches, I was able to define ADAP1 as an unexpected tuner of CD4+ T cell activation programs and co-opted by HIV-1 to escape latency.Item Functional Characterization of the HIV-1 NEF Acidic Cluster(2005-05-12) Baugh, Laura Louise; Garcia-Martinez, J. VictorThe human immunodeficiency virus (HIV) infects over 39.5 million people worldwide (UNAIDS/WHO 2006 Epidemic Report). Unfortunately, various socioeconomic factors have hastened the epidemic spread of HIV in Africa and in third world countries where many individuals do not have access to appropriate healthcare. Vaccines have, as of yet, been unsuccessful as a preventive measure; however, new measures, including peptide inhibitors and microbicides, are currently being developed and studied for their efficacy. The potential of finding new targets for controlling or preventing infection relies heavily on developments relating to the basic science of the virus as well as the host environment. HIV and simian immunodeficiency virus (SIV) encode an accessory protein Nef proposed to promote pathogenesis based on evidence from HIV-infected longterm nonprogressors and SIV studies in non-human primates. The pathogenic potential of Nef has been studied in relation to some of its in vitro effects including: (1) the downregulation of major histocompatibility complex I (MHC-I), (2) the downregulation of CD4, (3) infectivity enhancement, and (4) Pak2 activation. These in vitro effects, with the exception of CD4 downregulation, rely in part upon Nef's acidic cluster which has been reported to affect Nef's redirection of cell surface MHC-I to the trans-Golgi network by binding to the cytosolic adapter phosphofurin acidic cluster sorting-1 (PACS-1) (19, 147). Because Nef is potentially significant to the course of disease progression, correlations between the amino acid composition of the Nef acidic cluster and Nef activity were characterized. Nef acidic cluster mutants reducing the acidic residues in the cluster from 4 to 2, 1, and 0 were developed. Phenotypes for these mutants indicate at least one or more of the four acidic cluster glutamates are involved not only in MHC-I downregulation, but also Pak2 activation and infectivity enhancement. However, because partial activity is observed with one acidic residue and full activity with only two acidic residues, these data lead to the following conclusion: Although the Nef acidic cluster contributes to a conformational integrity important for Nef-mediated MHC-I downregulation as well as other Nef activities, it is not a prototypical acidic cluster determinant involved in PACS-1-mediated trafficking.Item PI(4)P-Dependent Recruitment of Clathrin Adaptors to the Trans-Golgi Network(2005-04-29) Wang, Jing; Yin, Helen L.The Trans Golgi Network (TGN) is the cell's central sorting station, and the complex trafficking patterns are organized by many types of trafficking adaptors. These include the heterotetrameric adaptor protein complexes (APs) and the monomeric Golgi-localized, gamma-ear containing, Arf-binding proteins (GGAs). The fundamental question of how these adaptors are recruited to TGN membrane remains unclear. Previous studies have shown that adaptor recruitment to the TGN is absolutely dependent on the small GTPase ADP ribosylation factor 1 (Arf1), but paradoxically, Arf1 has a broader intracellular distribution than these adaptors. We found that the Golgi is particularly enriched in phosphatidylinositol 4 phosphate [PI(4)P] and that the clathrin adaptor AP-1 binds PI(4)P directly, suggesting that PI(4)P binding may specify the TGN-specific recruitment in conjunction with Arf1. My studies showed that another monomeric clathrin adaptor GGA also binds PI(4)P and Arf1 independently. The C-terminal "triple helix bundle" of the GGA GAT domain is a polyfunctional module that interacts with multiple partners including PI(4)P and ubiquitin, and ubiquitin may provide a recognition signal for GGAs to control protein sorting. We found that PI(4)P increases wild type GAT binding to ubiquitin-conjugated agarose beads, but has no effect on a mutant GAT that does not bind PI(4)P. Therefore, PI(4)P may be an allosteric regulator of GGAs which enhances ubiquitin binding to GGAs. Based on these results, we conclude: (1) PI(4)P defines the TGN organelle identity by recruiting TGN-targeted adaptors; (2) TGN-enriched adaptors are recruited to the Golgi by binding to both PI(4)P and Arf1, and neither alone is sufficient; (3) PI(4)P acts as a scaffold, and may also be an allosteric regulator for GGAs that modulates GGA function with other ligands. We propose that the integration of combinatorial inputs from PI(4)P, Arf1 and ubiquitin may coordinately specify clathrin adaptor TGN recruitment through multiple low-affinity interactions.