A Single Cell Perspective of the Effects of Dopamine in the Regulation of HIV Latency Phenotypes in a Myeloid Cell Model
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE278043 | GEO | 2024/12/25
REPOSITORIES: GEO
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Project description:Effects of dopamine on HIV latently-infected U1 macrophage cell line
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Project description:Purpose: This study aimed to identify potential biomarkers related to HIV latency on the cell surface. Experimental design: A subcellular proteomic study was performed, using an HIV-1 latency cell line (U1 (HIV-1-integrated U937 cell line)) and control (U937). Plasma membrane proteins were enriched, digested, labeled using an iTRAQ kit, and identified by LC-MS. Differentially expressed proteins were analyzed using bioinformatics technology, followed by western blot validation in cell lines and resting CD4+ T cells from patients. Results: Totally, 110 differentially expressed proteins were identified in U1 compared with U937. Bioinformatics analysis suggested that the immune response and phagosomes were associated with the altered proteins, and HIV proteins such as Vpr were involved in the regulation of these differential proteins. Lysosome-associated membrane glycoprotein 2 (LAMP2), complement decay-accelerating factor, integrin alpha-6, and leukocyte surface antigen CD47 were shown to be downregulated in HIV-1 latent cell lines. Of these, LAMP2 was also shown to be downregulated in enriched resting CD4+ T cells from HIV-infected patients compared with those from healthy volunteers. Conclusions and clinical relevance: Our results indicate that HIV might decrease immunity proteins such as LAMP2 and CD47 to avoid immune recognition. LAMP2 may be a potential biomarker for HIV-1 latency.
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Project description:G2019S mutaion of LRRK2 is known to increase mRNA translation. We perform ribosome profiling to study defective translation using human dopamine neuron models. Patient-derived human dopamine neurons with G2019S LRRK2 mutation were generated and used. Also a mutation-corrected isogenic pair line was used.
2018-07-01 | GSE90469 | GEO
Project description:The dopamine transporter facilitates dopamine reuptake from the extracellular space to terminate neurotransmission. The transporter belongs to the neurotransmitter:sodium symporter family, which includes transporters for serotonin, norepinephrine, and GABA that utilize the Na+ gradient to drive the uptake of substrate. Decades ago, it was shown that the serotonin transporter also antiports K+, but investigations of K+-coupled transport in other neurotransmitter:sodium symporters have been inconclusive. Here, we show that ligand binding to the drosophila- and human dopamine transporters are inhibited by K+, and the conformational dynamics of the drosophila dopamine transporter in K+ are divergent from the apo- and Na+-states. Furthermore, we found that K+ increased dopamine uptake by the drosophila dopamine transporter in liposomes, and visualized Na+ and K+ fluxes in single proteoliposomes using fluorescent ion indicators. Our results expand on the fundamentals of dopamine transport and prompt a reevaluation of the impact of K+ on other transporters in this pharmacologically important family.
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Project description:Cells: ACH-2, A3.01, J1.1, and U1 cells were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. U-937 cells were obtained from American Type Culture Collection (Manassas, VA). ACH-2, J1.1 and U1 are chronically infected cell lines harboring HIV-1 LAV strain, while A3.01, Jurkat, and U-937 are the corresponding parental uninfected cell lines. Cells were grown in RPMI-1640 (Invitrogen, San Diego, CA) with 10% fetal bovine serum (FBS, Invitrogen), 5% penicillin-streptomycin (Invitrogen), and 2mM glutamine (Invitrogen). Cells were maintained at a concentration of 1x10e6 cells/ml in T-175 flasks. Cell concentrations and cell viability were monitored throughout the experiment. Cells were harvested by centrifugation at 1000 rpm for 10 minutes. Harvested cells were washed thrice with ice-cold PBS to remove media; cell pellets were snap frozen using ethanol-dry ice mixture and stored at -80°C for subsequent RNA extraction. In order to compare expression profiles of chronically infected cell lines, ACH-2, U1, and J1.1 and their uninfected parental cell lines, A3.01, U-937 and Jurkat cells respectively, were grown under identical conditions but in presence of AZT (250 nM) in growth media and cells were harvested as described above. In these studies, no inducing agent was used in either chronically infected or uninfected parental cell lines so as to study changes in cellular gene expression cells latently infected with HIV and uninfected cells. Total RNA Extraction: Total RNA was extracted using RNEasy Midiprep Kits per manufacturer's protocol (Qiagen, Valencia, CA). RNA concentrations and purity were measured by spectrophotometry, RNA quality (absence of RNA degradation) was assessed by gel electrophoresis. RNA concentration was adjusted to the levels required for subsequent microarray experiment protocols by concentration in a SpeedVac (Savant Instruments, Holbrook, CA). RNA samples (6-7 µg/µL) were stored in 100 µL TE buffer at -80°C. Microarray Studies: Total RNA obtained from chronically infected and corresponding uninfected parental cells were used for microarray experiments. For each cell line, RNA from the chronically infected cells (ACH-2, J1.1 or U1)and RNA from the corresponding induced, uninfected cells (A3.01 , Jurkat or U937) were compared on the same array. Microarrays were obtained from the National Cancer Institute Microarray Facility, Advanced Technology Center (Gaithersburg, MD). The microarrays (Hs. UniGem2) contained 10,368 cDNA spots on each glass slide. The cDNAs were selected for spotting on the slides based on their known or probable involvement in oncogenesis, signal transduction, apoptosis, immune function, inflammatory pathways, cellular transport, transcription, protein translation and other important cellular functions. A number of expressed sequence tags (ESTs) from unknown genes homologous to known genes and cDNAs encoding housekeeping genes were also included in these gene sets. For each array, 50 µg of total RNA from chronically infected cells and 70 µg of total RNA from corresponding uninfected cells was labeled with Cy-3-dUTP and Cy-5-dUTP respectively. Higher amounts of RNA were used for Cy-5 labeling to minimize the disparities in dye incorporation. Following reverse transcription, the labeled cDNAs were then combined and purified using MicroCon YM-30 (Millipore, Bedford, MA) spin column filters, to remove any unincorporated nucleotides. 8-10 µg each of Cot-1 DNA, (Boehringer Mannheim, Indianapolis, IN), yeast tRNA (Sigma) and polyA (Amersham Biosciences) were added to the reaction mixture and heated at 100°C for 1 minute. Hybridization of the labeled cDNA to the microarray was carried out at 65°C overnight, followed by washes with 1X SSC, 0.2X SSC and 0.05X SSC respectively. The slides were dried by centrifugation at 1000 rpm for 3 minutes and then scanned as described below. Microarray Scanning and Data Analysis: The slides were scanned using an Axon GenePix 4000 scanner (Axon Instruments, Union City, CA). The photomultiplier tube values (PMT) were adjusted to obtain equivalent intensities at both wavelengths used, 635 nm and 532 nm for the Cy5 and Cy3 channels respectively. Image analysis was performed using GenePix analysis software (Axon Instruments) and data analysis was performed using the microArray Database (mAdb) system hosted by the Center for Information Technology and Center for Cancer Research at NIH (http://nciarray.nci.nih.gov/). Keywords = HIV Keywords = Latency
2010-06-06 | E-GEOD-1443 | biostudies-arrayexpress
Project description:The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe the global Na+- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na+- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.
2019-11-12 | PXD013841 | Pride
Project description:Impaired dopamine homeostasis is an early event in the pathogenesis of Parkinson's disease. Generation of intracellular reactive oxygen species consequent to dopamine oxidation leads to mitochondrial dysfunction and eventually cell death. Alterations in the mitochondrial proteome due to dopamine exposure were investigated in the SH-SY5Y human neuroblastoma cell line. The combination of two orthogonal proteomic approaches, two-dimensional electrophoresis and shotgun proteomics, was used to highlight the specific pathways perturbed by the increase of intracellular dopamine, in comparison with those perturbed by a specific mitochondrial toxin (4-methyphenylpyridinium, MPP+), a neurotoxin causing Parkinsonism-like symptoms in animal models. Proteins altered by MPP+ did not completely overlap with those affected by dopamine treatment. In particular, the MPP+ target complex I component NADH dehydrogenase [ubiquinone] iron-sulfur protein 3 was not affected by dopamine together with 26 other proteins. The comparison of proteomics approaches highlighted the fragmentation of some mitochondrial proteins, suggesting an alteration of the mitochondrial protease activity. Pathway and disease association analysis of the proteins affected by dopamine revealed the overrepresentation of the Parkinson's disease and the parkin-ubiquitin proteasomal system pathways and of gene ontologies associated to generation of precursor metabolites and energy, response to topologically incorrect proteins and programmed cell death. These alterations may be globally interpreted in part as the result of a direct effect of dopamine on mitochondria (e.g. alteration of the mitochondrial protease activity) and in part as the effect on mitochondria of a general activation of cellular processes (e.g. regulation of programmed cell death).
2014-04-15 | PXD000838 | Pride
Project description:Dopaminylation, the covalent attachment of dopamine to the side chain of glutamine (Gln, Q) in proteins, has been identified as a class of posttranslational modification. Due to the limited number of substrates, the functions and underlying molecular mechanisms of dopaminylation are not fully characterized. Utilizing an alkyne-functionalized dopamine probe, we have developed a method to selectively enrich dopaminylated proteins in the whole-cell context. We identified 4133 proteins potentially modified with dopamine and validated the modification of histone H4 glutamine 27 by dopamine (H4Q27dop). H4Q27dop can inhibit cell proliferation through downregulating cyclin D1 gene CCND1 transcription, a classical well-known proliferation promoter. Our study provides a valuable resource of putative substrate proteins modified with dopamine and reveals a novel mechanism of dopamine regulating cell growth in a neuroblastoma cancer model.
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