RESUMEN
Protein phosphatase 1 (PP1), a cellular serine/threonine phosphatase, is targeted to cellular promoters by its major regulatory subunits, PP1 nuclear targeting subunit, nuclear inhibitor of PP1 (NIPP1) and RepoMan. PP1 is also targeted to RNA polymerase II (RNAPII) by NIPP1 where it can dephosphorylate RNAPII and cycle-dependent kinase 9 (CDK9). Here, we show that treatment of cells with a small molecule activator of PP1 increases the abundance of a neuregulin-1 (NRG-1)-derived peptide. NRG-1 mRNA and protein levels were increased in the cells stably or transiently expressing mutant NIPP1 (mNIPP1) that does not bind PP1, but not in the cells expressing NIPP1. Expression of mNIPP1 also activated the NRG-1 promoter in an NF-κB-dependent manner. Analysis of extracts from mNIPP1 expressing cells by glycerol gradient centrifugation showed a redistribution of PP1 and CDK9 between large and small molecular weight complexes, and increased CDK9 Thr-186 phosphorylation. This correlated with the increased CDK9 activity. Further, RNAPII co-precipitated with mNIPP1, and phosphorylation of RNAPII C-terminal domain (CTD) Ser-2 residues was greater in cells expressing mNIPP1. In mNIPP1 expressing cells, okadaic acid, a cell-permeable inhibitor of PP1, did not increase Ser-2 CTD phosphorylation inhibited by flavopiridol, in contrast to the NIPP1 expressing cells, suggesting that PP1 was no longer involved in RNAPII dephosphorylation. Finally, media conditioned with mNIPP1 cells induced the proliferation of wild type 84-31 cells, consistent with a role of neuregulin-1 as a growth promoting factor. Our study indicates that deregulation of PP1/NIPP1 holoenzyme activates NRG-1 expression through RNAPII and CDK9 phosphorylation in a NF-κB dependent manner.
RESUMEN
Highly active antiretroviral (ARV) therapy (HAART) for chronic suppression of HIV replication has revolutionized the treatment of HIV/AIDS. HAART is no panacea; treatments must be maintained for life. Although great progress has been made in ARV therapy, HIV continues to replicate in anatomical and intracellular sites where ARV drugs have restricted access. Nanotechnology has been considered a platform to circumvent some of the challenges in HIV/AIDS treatment. Dispersion polymerization was used to fabricate two types (PMM and ECA) of polymeric nanoparticles, and each was successfully loaded with four ARV drugs (zidovudine, lamivudine, nevirapine, and raltegravir), followed by physicochemical characterization: scanning electron microscope, particle size, zeta potential, drug loading, and in vitro availability. These nanoparticles efficiently inhibited HIV-1 infection in CEM T cells and peripheral blood mononuclear cells; they hold promise for the treatment of HIV/AIDS. The ARV-loaded nanoparticles with polyethylene glycol on the corona may facilitate tethering ligands for targeting specific receptors expressed on the cells of HIV reservoirs.
RESUMEN
Complete eradication of HIV-1 infection is impeded by the existence of latent HIV-1 reservoirs in which the integrated HIV-1 provirus is transcriptionally inactive. Activation of HIV-1 transcription requires the viral Tat protein and host cell factors, including protein phosphatase-1 (PP1). We previously developed a library of small compounds that targeted PP1 and identified a compound, SMAPP1, which induced HIV-1 transcription. However, this compound has a limited bioavailability in vivo and may not be able to reach HIV-1-infected cells and induce HIV-1 transcription in patients. We packaged SMAPP1 in polymeric polyethylene glycol polymethyl methacrylate nanoparticles and analyzed its release and the effect on HIV-1 transcription in a cell culture. SMAPP1 was efficiently packaged in the nanoparticles and released during a 120-hr period. Treatment of the HIV-1-infected cells with the SMAPP1-loaded nanoparticles induced HIV-1 transcription. Thus, nanoparticles loaded with HIV-1-targeting compounds might be useful for future anti-HIV-1 therapeutics.