The AAA+ family in eukaryotes has many members in various cellular compartments with a role in protein unfolding and degradation. We show that the mitochondrial AAA-ATPase Bcs1 has an unusual ...function in protein translocation. Bcs1 mediates topogenesis of the Rieske protein, Rip1, a component of respiratory chains in bacteria, mitochondria, and chloroplasts. The oligomeric AAA-ATPase Bcs1 is involved in export of the folded Fe-S domain of Rip1 across the inner membrane and insertion of its transmembrane segment into an assembly intermediate of the cytochrome
bc
1 complex, thus revealing an unexpected mechanistical concept of protein translocation across membranes. Furthermore, we describe structural elements of Rip1 required for recognition and export by as well as ATP-dependent lateral release from the AAA-ATPase. In bacteria and chloroplasts Rip1 uses the Tat machinery for topogenesis; however, mitochondria have lost this machinery during evolution and a member of the AAA-ATPase family has taken over its function.
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► An unusual function for a member of the AAA-ATPase family ► Translocation of folded proteins across the mitochondrial inner membrane ► Evolution of mitochondria: replacement of the Tat system by the BCS1 complex
•VIP-TAT has increased traversing efficiency.•VIP-TAT inhibited scopolamine induced amnesia more effectively than VIP.•VIP-TAT had higher affinity for PAC1.1Co-first author.
A novel peptide VIP-TAT ...with a cell penetrating peptide TAT at the C-terminus of VIP was constructed and prepared using intein mediated purification with an affinity chitin-binding tag (IMPACT) system to enhance the brain uptake efficiency for the medical application in central nervous system. It was found by labeling VIP-TAT and VIP with fluorescein isothiocyanate (FITC) that the extension with TAT increased the brain uptake efficiency of VIP-TAT significantly. Then short-term and long-term treatment with scopolamine (Scop) was used to evaluate the effect of VIP-TAT or VIP on Scop induced amnesia. Both short-term and long-term administration of VIP-TAT inhibited the latent time reduction in step-through test induced by Scop significantly, but long-term administration of VIP aggravated the Scop induced amnesia. Long-term i.p. injection of VIP-TAT was shown to have positive effect by inhibiting the oxidative damage, apoptosis and the cholinergic system activity reduction that induced by Scop, while VIP exerted negative effect in brain opposite to that in periphery system. The in vitro data showed that VIP-TAT had not only protective but also proliferative effect on Neuro2a cells which was inhibited by PAC1 antagonist PACAP(6-38). Competition binding assay and cAMP assay confirmed that VIP-TAT had higher affinity and activation for PAC1 than VIP. So it was concluded that the significantly stronger protective effect of VIP-TAT against Scop induced amnesia than VIP was due to (1) the enhanced brain uptake efficiency of VIP-TAT and (2) the increased affinity and activation of VIP-TAT for receptor PAC1.
The recombinant HIV-1 CRF02_AG is prevalent in West-Central Africa but its effects on the blood-brain barrier (BBB) and HIV-associated neurocognitive disorders (HAND) are not known. We analyzed the ...effects of Tat from HIV-1 subtype-B (Tat.B) and CRF02_AG (Tat.AG) on primary human brain microvascular endothelial cells (HBMEC), the major BBB component. Exposure of HBMEC to Tat.B increased IL-6 expression and transcription by 9- (
P
< 0.001) and 113-fold (
P
< 0.001), respectively, whereas Tat.AG increased IL-6 expression and transcription by 2.7–3.8-fold and 35.7-fold (
P
< 0.001), respectively. Tat.B induced IL-6 through the interleukin-1 receptor-associated kinase (IRAK)-1/4/mitogen-activated protein kinase kinase(MKK)/C-jun N-terminal kinase(JNK) pathways, in an activator protein-1(AP1)- and nuclear factor-kappaB (NFκB)-independent manner, whereas Tat.AG effects occurred via MKK/JNK/AP1/NFκB pathways. Tat-induced effects were associated with activation of c-jun (serine-63) and SAPK/JNK (Thr183/Tyr185). We demonstrated increased expression of transcription factors associated with these pathways (Jun, RELB, CEBPA), with higher levels in Tat.B-treated cells compared to Tat.AG. Functional studies showed that Tat.B and Tat.AG decreased the expression of tight junction proteins claudin-5 and ZO-1 and decreased the trans-endothelial electric resistance (TEER); Tat.B induced greater reduction in TEER, claudin-5, and ZO-1, compared to Tat.AG. Overall, our data showed increased inflammation and BBB dysfunction with Tat.B, compared to Tat.AG. This suggests these two HIV-1 subtypes differentially affect the BBB and central nervous system; our data provides novel insights into the molecular basis of these differential Tat-mediated effects.
Cell-penetrating peptides (CPPs), also referred to as protein transduction domains (PTDs), can mediate the cellular uptake of a wide range of macromolecules including peptides, proteins, ...oligonucleotides, and nanoparticles, and thus have received considerable attention as a promising method for drug delivery in vivo. Here, we report that CPP/PTDs facilitate the extravasation of fused proteins by binding to neuropilin-1 (NRP1), a vascular endothelial growth factor (VEGF) co-receptor expressed on the surface of endothelial and some tumor cells. In this study, we examined the capacity of the amphipathic and cationic CPP/PTDs, PTD-3 and TAT-PTD, respectively, to bind cells in vitro and accumulate in xenograft tumors in vivo. Notably, these functions were significantly suppressed by pre-treatment with NRP1-neutralizing Ab. Furthermore, co-injection of iRGD, a cyclic peptide known to increase NRP1-dependent vascular permeability, significantly reduced CPP/PTD tumor delivery. This data demonstrates a mechanism by which NRP1 promotes the extravasation of CPP/PTDs that may open new avenues for the development of more efficient CPP/PTD delivery systems.
We provide a detailed study of the oxide- semiconductor interface trap assisted tunneling (TAT) mechanism in tunnel FETs to show how it contributes a major leakage current path before the ...band-to-band tunneling (BTBT) is initiated. With a modified Shockley-Read-Hall formalism, we show that at room temperature, the phonon assisted TAT current always dominates and obscures the steep turn ON of the BTBT current for common densities of traps. Our results are applicable to top gate, double gate, and gate-all-around structures, where the traps are positioned between the source-channel tunneling regions. Since the TAT has strong dependence on electric field, any effort to increase the BTBT current by enhancing local electric field also increases the leakage current. Unless the BTBT current can be increased separately, calculations show that the trap density Dit has to be decreased by 40-100 times compared with the state of the art in order for the steep turn ON (for III-V materials) to be clearly observable at room temperature. We find that the combination of the intrinsic sharpness of the band edges (Urbach tail) and the surface trap density determines the subthreshold swing.
Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) protein is widely expressed in the brain and it is known that this protein is involved in cell survival including dopaminergic neuronal cells. ...Oxidative stress is known as one of the major causes of degenerative diseases including ischemia. In this study, we investigated the effect of CIAPIN1 protein on hippocampal neuronal (HT-22) cell damage induced by hydrogen peroxide (H2O2) and in an animal model of ischemia using Tat-CIAPIN1 fusion protein which can transduce into cells. Tat-CIAPIN1 protein transduced into HT-22 cells and significantly inhibited cell death, DNA fragmentation, and reactive oxygen species (ROS) generation. Also, Tat-CIAPIN1 protein enhances cell survival via the regulation of Akt, MAPK, NF-κB and apoptotic signaling pathways in the H2O2 treated cells. In an ischemic animal model, Tat-CIAPIN1 protein transduced into the brain and protected neuronal cell death of hippocampal CA1 region induced by ischemic insult. In conclusion, we demonstrated that Tat-CIAPIN1 protein has protective effects against hippocampal neuronal cell damage induced by ischemic injury, suggesting that Tat-CIAPIN1 protein may provide a potential therapeutic agent for ischemia.
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•Tat-CIAPIN1 transduces into HT-22 cells and animal brain.•Transduced Tat-CIAPIN1 protects against H2O2-induced cellular toxicity.•Tat-CIAPIN1 regulates MAPK and apoptosis signaling pathways.•Tat-CIAPIN1 protects against neuronal cell death in an ischemic animal model.•Tat-CIAPIN1 can be a therapeutic agent for neuronal diseases including ischemia.
In this work, a novel protonable copolymer was designed to deliver siRNA through the inhalation route, as an innovative formulation for the management of asthma. This polycation was synthesized by ...derivatization of α,β-poly(N-2-hydroxyethyl)D,L-aspartamide (PHEA) first with 1,2-Bis(3-aminopropylamino)ethane (bAPAE) and then with a proper amount of maleimide terminated poly(ethylene glycol) (PEG-MLB), with the aim to increase the superficial hydrophilicity of the system, allowing the diffusion trough the mucus layer. Once the complexation ability of the copolymer has been evaluated, obtaining nanosized polyplexes, polyplexes were functionalized on the surface with a thiolated TAT peptide, a cell-penetrating peptide (CPP), exploiting a thiol-ene reaction. TAT decorated polyplexes result to be highly cytocompatible and able to retain the siRNA with a suitable complexation weight ratio during the diffusion process through the mucus. Despite polyplexes establish weak bonds with the mucin chains, these can diffuse efficiently through the mucin layer and therefore potentially able to reach the bronchial epithelium. Furthermore, through cellular uptake studies, it was possible to observe how the obtained polyplexes penetrate effectively in the cytoplasm of bronchial epithelial cells, where they can reduce IL-8 gene expression, after LPS exposure. In the end, in order to obtain a formulation administrable as an inhalable dry powder, polyplexes were encapsulated in mannitol-based microparticles, by spray freeze drying, obtaining highly porous particles with proper technological characteristics that make them potentially administrable by inhalation route.
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Despite the effectiveness of combined anti-retroviral therapy, human immunodeficiency virus (HIV) infected-patients frequently report diarrhea and neuropsychological deficits. It is claimed that the ...viral HIV-1 Trans activating factor (HIV-1 Tat) protein is responsible for both diarrhea and neurotoxic effects, but the underlying mechanisms are not known. We hypothesize that colonic application of HIV-1 Tat activates glial cells of the enteric nervous system (EGCs), leading to a neuroinflammatory response able to propagate to the central nervous system. We demonstrated that HIV-1 Tat-induced diarrhea was associated with a significant activation of glial cells within the colonic wall, the spinal cord and the frontal cortex, and caused a consistent impairment of the cognitive performances. The inhibition of glial cells activity by lidocaine, completely abolished the above-described effects. These observations point out the role of glial cells as putative effectors in HIV-1 Tat-associated gastrointestinal and neurological manifestations and key regulators of gut-brain signaling.
A high level of low‐density lipoprotein cholesterol (LDL‐C) in the blood is a major risk factor for coronary heart disease. Herein, we present a triple‐targeting strategy to generate a ...loss‐of‐function mutation in Pcsk9, which regulates plasma cholesterol levels, using a nanocarrier‐delivered CRISPR/Cas9 system. Nuclear localization signal (NLS)‐tagged Cas9 and Pcsk9‐targeted single guide RNA (sgPcsk9) were complexed with gold nanoclusters (GNCs) modified with cationic HIV‐1‐transactivating transcriptor (TAT) peptide and further encapsulated in a galactose‐modified lipid layer to target the nanoclusters to the liver. The resulting nanoclusters had an in vitro Pcsk9‐editing efficiency of about 60 % and resulting in a decrease in plasma LDL‐C in mice of approximately 30%. No off‐target mutagenesis was detected in 10 sites with high similarity. This approach may have therapeutic potential for the prevention and treatment of cardiovascular disease without side effects.
A gold‐nanocluster‐based CRISPR/Cas9 delivery system could deliver CRISPR/Cas9 to the liver of mice, mutating the Pcsk9 gene and decreasing low‐density lipoprotein cholesterol (LDL‐C) levels in the plasma. This approach uses galactose to target the asialoglycoprotein receptor (ASGPR) on hepatocytes, and the HIV TAT peptide and a nuclear localization signal to target nuclei, and may be useful for the prevention and treatment of cardiovascular disease.
•HIV-1 Tat exposure disrupted the BBB, as evidenced by Na-F and HRP leakage into the brain.•HIV-1 Tat exposure increased phagocytic macrophages/microglia within the caudate/putamen.•HIV-1 Tat ...activated both perivascular and tissue-resident macrophages/microglia.
HIV-1 infection results in blood-brain barrier (BBB) disruption, which acts as a rate-limiting step for HIV-1 entry into the CNS and for subsequent neuroinflammatory/neurotoxic actions. One mechanism by which HIV may destabilize the BBB involves actions of the HIV-1 regulatory protein, trans-activator of transcription (Tat). We utilized a conditional, Tat-expressing transgenic murine model to examine the influence of Tat1-86 expression on BBB integrity and to assess the relative numbers of phagocytic perivascular macrophages and microglia within the CNS in vivo. The effects of Tat exposure on sodium-fluorescein (Na-F; 0.376kDa), horseradish peroxidase (HRP; 44kDa), and Texas Red-labeled dextran (70kDa) leakage into the brain were assessed in Tat-exposed (Tat+) and control (Tat−) mice. Exposure to HIV-1 Tat significantly increased both Na-F and HRP, but not the larger sized Texas Red-labeled dextran, confirming BBB breakdown and also suggesting the breach was limited to molecules <70kDa. Additionally, at 5 d after Tat induction, Alexa Fluor® 488-labeled dextran was bilaterally infused into the lateral ventricles 5 d before the termination of the experiment. Within the caudate/putamen, Tat induction increased the proportion of dextran-labeled Iba-1+ phagocytic perivascular macrophages (∼5-fold) and microglia (∼3-fold) compared to Tat− mice. These data suggest that HIV-1 Tat exposure is sufficient to destabilize BBB integrity and to increase the presence of activated, phagocytic, perivascular macrophages and microglia in an in vivo model of neuroAIDS.
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