HIV gene expression is modulated by the combinatorial activity of the HIV transcriptional activator, Tat, host transcription factors, and chromatin remodeling complexes. To identify host factors ...regulating HIV transcription, we used specific single-guide RNAs and endonuclease-deficient Cas9 to perform chromatin affinity purification of the integrated HIV promoter followed by mass spectrometry. The scaffold protein, p32, also called ASF/SF2 splicing factor-associated protein, was identified among the top enriched factors present in actively transcribing HIV promoters but absent in silenced ones. Chromatin immunoprecipitation analysis confirmed the presence of p32 on active HIV promoters and its enhanced recruitment by Tat. HIV uses Tat to efficiently recruit positive transcription elongation factor b (p-TEFb) (CDK9/CCNT1) to TAR, an RNA secondary structure that forms from the first 59 bp of HIV transcripts, to enhance RNAPII transcriptional elongation. The RNA interference of p32 significantly reduced HIV transcription in primary CD4
T cells and in HIV chronically infected cells, independently of either HIV splicing or p32 anti-splicing activity. Conversely, overexpression of p32 specifically increased Tat-dependent HIV transcription. p32 was found to directly interact with Tat's basic domain enhancing Tat stability and half-life. Conversely, p32 associates with Tat via N- and C-terminal domains. Likely due its scaffold properties, p32 also promoted Tat association with TAR, p-TEFb, and RNAPII enhancing Tat-dependent HIV transcription. In sum, we identified p32 as a host factor that interacts with and stabilizes Tat protein, promotes Tat-dependent transcriptional regulation, and may be explored for HIV-targeted transcriptional inhibition.
The human autophagy-related protein ATG7 (hATG7), an E1-like ubiquitin enzyme, activates two ubiquitin-like proteins, LC3 (Atg8) and Atg12, and promotes autophagosome formation. While hATG7 plays an ...essential role for the autophagy conjugation system, the production of full-length functional hATG7 in bacterial systems remains challenging. Previous studies have demonstrated that the HIV-1 virus-encoded Tat peptide (‘GRKKRRQRRR’) can increase the yield and solubility of heterologous proteins. Here, functional full-length hATG7 was expressed using the pET28b-Tat expression vector in the Escherichia coli BL21 (DE3) strain. Recombinant hATG7 protein aggregated as inclusion bodies while expressed with widely used prokaryotic expression plasmids. In contrast, the solubility of Tat-tagged hATG7 increased significantly with prolonged time compared to Tat-free hATG7. The recombinant proteins were purified to >90% homogeneity under native conditions with a single step of affinity chromatography purification. The results of in vitro pull-down and LC3B–I lipidation assays showed that Tat-tagged hATG7 directly interacted with LC3B–I and promoted LC3B–I lipidation, suggesting that Tat-tagged hATG7 has significant catalytic activity. Overall, this study provides a novel method for improving the functional expression of full-length hATG7 in bacterial systems by fusion with the Tat peptide, a process which may be applied in future studies of hATG7 structure and function.
•Functional overexpression of recombinant full-length human ATG7 in Escherichia coli.•The recombinant protein showed high purity and excellent biological function.•Tat peptide (‘GRKKRRQRRR’) could increase the solubilities of human ATG7.
Highlights • TAT expression in the brain enhances sensitivity to methamphetamine. • TAT expression decreased striatal dopamine receptors expression. • TAT expression and methamphetamine increased ...recruitment of midbrain dopamine neurons. • TAT expression and methamphetamine differentially modulated adenosine receptors expression. • TAT-induced neuroadaptations may contribute to comorbid methamphetamine abuse and HIV.
Precisely controlling the interaction of nanoparticles with biological systems (nanobio interactions) from the injection site to biological targets shows great potential for biomedical applications. ...Inspired by the ability of nanoparticles to alter their physicochemical properties according to different stimuli, we explored the tumor acidity and near-infrared (NIR) light activated transformable nanoparticle DATAT-NPIR&DOX. This nanoparticle consists of a tumor acidity-activated TAT the TAT lysine residues’ amines was modified with 2,3-dimethylmaleic anhydride (DA), a flexible chain polyphosphoester core coencapsulated a NIR dye IR-780, and DOX (doxorubicin). The physicochemical properties of the nanoparticle can be controlled in a stepwise fashion using tumor acidity and NIR light, resulting in adjustable nanobio interactions. The resulting transformable nanoparticle DATAT-NPIR&DOX efficiently avoids the interaction with mononuclear phagocyte system (MPS) (“stealth” state) due to the masking of the TAT peptide during blood circulation. Once it has accumulated in the tumor tissues, DATAT-NPIR&DOX is reactivated by tumor acidity and transformed into the “recognize” state in order to promote interaction with tumor cells and enhance cellular internalization. Then, this nanoparticle is transformed into “attack” state under NIR irradiation, achieving the supersensitive DOX release from the flexible chain polyphosphoester core in order to increase the DOX–DNA interaction. This concept provides new avenues for the creation of transformable drug delivery systems that have the ability to control nanobio interactions.
The widespread use of combinational antiretroviral therapies (cART) in developed countries has changed the course of Human Immunodeficiency Virus (HIV) infection from an almost universally fatal ...disease to a chronic infection for the majority of individuals. Although cART has reduced the severity of neurological damage in HIV-infected individuals, the likelihood of cognitive impairment increases with age, and duration of infection. As cART does not suppress the expression of HIV non-structural proteins, it has been proposed that a constitutive production of HIV regulatory proteins in infected brain cells may contribute to neurological damage. However, this assumption has never been experimentally tested. Here we take advantage of the leaky tetracycline promoter system in the Tat-transgenic mouse to show that a chronic very low-level expression of Tat is associated with astrocyte activation, inflammatory cytokine expression, ceramide accumulation, reductions in brain volume, synaptic, and axonal damage that occurs over a time frame of 1 year. These data suggest that a chronic low-level production of Tat may contribute to progressive neurological damage in virally suppressed HIV-infected individuals.
Abstract Delivery of therapeutic molecules to the brain for the treatment of Neurodegenerative diseases (ND) is a challenging task. This manuscript introduces a novel scheme of synthesizing ...peptide-tagged polyethylene glycol (PEG)ylated chitosan polymer to develop nanoparticles for siRNA delivery for use in ND. Specifically, this manuscript proposes a facile chemoselective conjugation of monomethoxy PEG, at the C2 hydroxyl group of chitosan polymer, with conjugation of PEG to a cell-penetrating peptide, Trans-Activator of Transcription. The synthesized Chitosan-PEG-TAT polymer was used to form the nanoparticles of approximately 5 nm, complexing siRNA to be delivered in neuronal cells (Neuro 2a), with no/minimal toxicity. The various intermediates and the final product formed during the synthesis were characterized using1 H Nuclear Magnetic Resonance and Fourier Transform Infrared Spectroscopy spectra. The morphological details of the nanoparticles were studied using Transmission Electron Microscopy. The nanoparticles were tested to deliver a functional siRNA against the Ataxin-1 gene in an in-vitro established model of a ND Spinocerebellar ataxia (SCA1) over-expressing ataxin protein. The results indicate successful suppression of the SCA1 protein following 48 h of transfection. Result of this study has potential in ND like SCA, Parkinson's, Alzheimer's and others.
Cell-penetrating peptides (CPPs), such as the HIV TAT peptide, are able to translocate across cellular membranes efficiently. A number of mechanisms, from direct entry to various endocytotic ...mechanisms (both receptor independent and receptor dependent), have been observed but how these specific amino acid sequences accomplish these effects is unknown. We show how CPP sequences can multiplex interactions with the membrane, the actin cytoskeleton, and cell-surface receptors to facilitate different translocation pathways under different conditions. Using "nunchuck" CPPs, we demonstrate that CPPs permeabilize membranes by generating topologically active saddle-splay ("negative Gaussian") membrane curvature through multidentate hydrogen bonding of lipid head groups. This requirement for negative Gaussian curvature constrains but underdetermines the amino acid content of CPPs. We observe that in most CPP sequences decreasing arginine content is offset by a simultaneous increase in lysine and hydrophobic content. Moreover, by densely organizing cationic residues while satisfying the above constraint, TAT peptide is able to combine cytoskeletal remodeling activity with membrane translocation activity. We show that the TAT peptide can induce structural changes reminiscent of macropinocytosis in actin-encapsulated giant vesicles without receptors.
Naturally occurring and synthetic short arginine containing protein transduction domains (PTDs), including HIV1 TAT, poly-Arg and Antp, have been used to deliver a wide variety of macromolecular, ...biologically active therapeutic cargo into cells, including peptides, proteins, antisense oligonucleotides and liposomes, in vitro and to treat pre-clinical models of cancer and stroke. PTDs enter cells in a rapid, receptor-independent fashion. Recently, large TAT-fusion proteins (in excess of 30,000 Da) were shown to transduce into cells by fluid-phase macropinocytosis, a specialized form of endocytosis that is independent of caveloe, clathrin and dynamin. However, it remains controversial as to whether or not PTD peptides (1000–5000 Da) enter cells via macropinocytosis and/or through an unknown alternative mechanism. Due to strong ionic interactions with the cell surface, previous measurements of PTD peptide internalization were inaccurate. Cationic PTD peptides containing variable numbers of arginine residues and conditions entered cells exclusively through macropinocytosis. In addition, no PTD peptide was found to enter cells at 4 °C, a long held assumption of transduction. Taken together, these observations provide a solid scientific basis for the development of novel biologically active transducible anticancer PTD peptide therapeutics.
Arbuscular mycorrhiza (AM) is an ecologically relevant symbiosis between most land plants and Glomeromycota fungi. The peculiar traits of AM fungi have so far limited traditional approaches such as ...genetic transformation. The aim of this work was to investigate whether the protein transduction domain of the HIV‐1 transactivator of transcription (TAT) protein, previously shown to act as a potent nanocarrier for macromolecule delivery in both animal and plant cells, may translocate protein cargoes into AM fungi. We evaluated the internalization into germinated spores of Gigaspora margarita of two recombinant TAT fusion proteins consisting of either a fluorescent (GFP) or a luminescent (aequorin) reporter linked to the TAT peptide. Both TAT‐fused proteins were found to enter AM fungal mycelia after a short incubation period (5–10 min). Ca²⁺ measurements in G. margarita mycelia pre‐incubated with TAT‐aequorin demonstrated the occurrence of changes in the intracellular free Ca²⁺ concentration in response to relevant stimuli, such as touch, cold, salinity, and strigolactones, symbiosis‐related plant signals. These data indicate that the cell‐penetrating properties of the TAT peptide can be used as an effective strategy for intracellularly delivering proteins of interest and shed new light on Ca²⁺ homeostasis and signalling in AM fungi.
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