Transcriptional Transactivation Research Articles

HIV-1 Tat amino acid residues that influence Tat-TAR binding affinity: a scoping review

HIV-1 remains a global health concern and to date, nearly 38 million people are living with HIV. The complexity of HIV-1 pathogenesis and its subsequent prevalence is influenced by several factors including the HIV-1 subtype. HIV-1 subtype variation extends to sequence variation in the amino acids of the HIV-1 viral proteins. Of particular interest is the transactivation of transcription (Tat) protein due to its key function in viral transcription. The Tat protein predominantly functions by binding to the transactivation response (TAR) RNA element to activate HIV-1 transcriptional elongation. Subtype-specific Tat protein sequence variation influences Tat-TAR binding affinity. Despite several studies investigating Tat-TAR binding, it is not clear which regions of the Tat protein and/or individual Tat amino acid residues may contribute to TAR binding affinity. We, therefore, conducted a scoping review on studies investigating Tat-TAR binding. We aimed to synthesize the published data to determine (1) the regions of the Tat protein that may be involved in TAR binding, (2) key Tat amino acids involved in TAR binding and (3) if Tat subtype-specific variation influences TAR binding. A total of thirteen studies met our inclusion criteria and the key findings were that (1) both N-terminal and C-terminal amino acids outside the basic domain (47–59) may be important in increasing Tat-TAR binding affinity, (2) substitution of the amino acids Lysine and Arginine (47–59) resulted in a reduction in binding affinity to TAR, and (3) none of the included studies have investigated Tat subtype-specific substitutions and therefore no commentary could be made regarding which subtype may have a higher Tat-TAR binding affinity. Future studies investigating Tat-TAR binding should therefore use full-length Tat proteins and compare subtype-specific variations. Studies of such a nature may help explain why we see differential pathogenesis and prevalence when comparing HIV-1 subtypes.

Abstract P5-14-02: Identifying oncogenic enhancer elements in TNBC of the Basal-like subtype using single-cell ATAC-seq and RNA-seq

Abstract Identification of the cis-regulatory elements controlling oncogenic transcriptional programs is critical to understanding tumor biology. To find cis-regulatory elements (i.e. gene enhancers) of oncogenic dependencies in Triple-Negative Breast Cancers (TNBC) of the Basal-like gene expression subtype, we generated matched single-cell transcriptome (scRNA-seq) and chromatin accessibility (scATAC-seq) profiles for two human Basal-like tumors and four normal mammary reduction samples. This unique dataset enabled us to correlate variations in chromatin structure with variations in gene expression revealing putative enhancers that are specifically active within cancer cells, but not within normal mammary ductal epithelial cells. We then leveraged the Cancer Dependency Map (DepMap) portal at the BROAD Institute to infer gene expression dependencies in breast cancer cell lines of the Basal-like molecular subtype. Putative cancer-specific enhancers were prioritized based on the transcriptional dependency of their target gene(s) in Basal-like cell lines as reported by the DepMap portal. Based on our preliminary analyses, we report several cancer-specific enhancers that drive the expression of important transcription factors such as EN1 and SOX4. These transcription factors are known to have profound effects on tumor biology, especially considering that high expression of EN1 is associated with brain metastasis and SOX4 is known to regulate immune evasion and PI3K/Akt signaling. Moreover, both of these transcription factors portend a worse outcome in TNBC patients. Thus, our analysis suggests that high levels of expression of these transcription factors is sustained specifically within the malignant cell types of these tumors, by the activity of these cancer-specific enhancers that are not typically active in normal epithelial cells. We are now performing CRISPR dCas9-KRAB experiments to epigenetically silence these cancer-specific enhancers and measure the consequences on expression of their predicted target genes. Additionally, we are investigating the trans-acting transcription factors that may physically bind to these enhancers to further regulate oncogenic transcription. By defining the regulatory logic of cancer cells at single-cell resolution, our work highlights the importance of cancer-specific and clinically relevant oncogenic regulatory elements in TNBC of the Basal-like subtype. Citation Format: Matthew J. Regner, Aatish Thennavan, Kamila Wisniewska, Susana Garcia-Recio, Raul Mendez-Giraldez, Philip Spanheimer, Charles M. Perou, Hector L. Franco. Identifying oncogenic enhancer elements in TNBC of the Basal-like subtype using single-cell ATAC-seq and RNA-seq [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-14-02.

Enhancing Methotrexate Delivery in the Brain by Mesoporous Silica Nanoparticles Functionalized with Cell-Penetrating Peptide using in Vivo and ex Vivo Monitoring.

The blood-brain barrier (BBB) acts as a physical/biochemical barrier that protects brain parenchyma from potential hazards exerted by different xenobiotics found in the systemic circulation. This barrier is created by "a lipophilic gate" as well as a series of highly organized influx/efflux mechanisms. The BBB bottleneck adversely affects the efficacy of chemotherapeutic agents in treating different CNS malignancies such as glioblastoma, an aggressive type of cancer affecting the brain. In the present study, mesoporous silica nanoparticles (MSNs) were conjugated with the transactivator of transcription (TAT) peptide, a cell-penetrating peptide, to produce MSN-NH-TAT with the aim of improving methotrexate (MTX) penetration into the brain. The TAT-modified nanosystem was characterized by Fourier transform infrared spectrometry (FTIR), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS), and N2 adsorption-desorption analysis. In vitro hemolysis and cell viability studies confirmed the biocompatibility of the MSN-based nanocarriers. In addition, in vivo studies showed that the MTX-loaded MSN-NH-TAT improved brain-to-plasma concentration ratio, brain uptake clearance, and the drug's blood terminal half-life, compared with the use of free MTX. Taken together, the results of the present study indicate that MSN functionalization with TAT is crucial for delivery of MTX into the brain. The present nanosystem represents a promising alternative drug carrier to deliver MTX into the brain via overcoming the BBB.

Neuroprotective effect of a novel brain-derived peptide, HIBDAP, against oxygen-glucose deprivation through inhibition of apoptosis in PC12 cells.

The effect of a novel brain-derived peptide,hypoxic-ischemic brain damage associated peptide (HIBDAP), on apoptosis after oxygen-glucose deprivation (OGD) in PC12 cells was investigated. The HIBDAP sequence (HSQFIGYPITLFVEKER) was coupled with the carrier peptide of the transactivator of transcription (TAT) sequence (YGRKKRRQRRR). FITC-labelled TAT-HIBDAP was observed by fluorescence microscopy. After TAT-HIBDAP treatment and OGD treatment, the PC12 cell apoptosis rate was analysed using lactate dehydrogenase (LDH) leakage and Annexin V-fluorescein isothiocyanate (FITC) assays. Mitochondrial membrane potential (ΔΨm) was examined by fluorescence microscopy. Protein expression of apoptotic factors was examined by Western blotting. FITC-labelled TAT-HIBDAP entered the PC12 cell nucleus. Compared with the OGD group, TAT-HIBDAP at low concentrations (1 µM, 5 µM, 10 µM) significantly reduced the apoptosis rate of PC12 cells (except at 20 µM); 5 µM TAT-HIBDAP had the most obvious effect. There were remarkable increases in ΔΨm at different concentrations (1 µM, 5 µM, 10 µM, 20 µM) of TAT-HIBDAP pretreatment, and 5 µM TAT-HIBDAP also had the most obvious effect. TAT-HIBDAP reversed the increased ratio of Bax/Bcl-2 and activation of Caspase-3 induced by OGD. TAT-HIBDAP is resistant to OGD-induced PC12 cell apoptosis by regulating the Bax/Bcl-2/Caspase-3 pathway, which may provide a novel therapeutic strategy for neonatal HIBD.

TAT Nanobody Exerts Antiviral Effect against PRRSV In Vitro by Targeting Viral Nucleocapsid Protein.

Porcine reproductive and respiratory syndrome (PRRS) is caused by the PRRS virus (PRRSV), which has brought huge economic losses to the pork industry worldwide since its first discovery in the late 1980s in North America. To date, there are no effective commercial vaccines or therapeutic drugs available for controlling the spread of PRRSV. Due to their unique advantages of high affinity and high specificity, nanobodies (Nbs) have received increasing attention in the process of disease diagnosis and treatment. Trans-activator transcription (TAT) can serve as a vector to carry specific proteins into cells by passing through cell membranes. In our previous study, a specific Nb against the PRRSV nucleocapsid (N) protein was screened using phage display technology. For this study, we developed a novel recombinant protein constituting a TAT-conjugated Nb, which we call TAT-Nb1. The target cell entry efficiency of TAT-Nb1 and its effect on PRRSV infection and replication were then investigated. Our results indicate that TAT delivered Nb1 into Marc-145 cells and porcine alveolar macrophages (PAMs) in a dose- and time-dependent manner. Furthermore, TAT-Nb1 dose-dependently suppressed PRRSV infection and replication, where this antiviral effect was independent of PRRSV strain. Co-immunoprecipitation results revealed that Nb1 efficiently interacted with the N protein of PRRSV. Taken together, the presented results suggest that TAT-Nb1 can effectively suppress PRRSV replication, and it may be considered as a new anti-PRRSV candidate drug.

An Effective Peptide-Based Platform for Efficient Exosomal Loading and Cellular Delivery of a microRNA.

Exosomes, membrane-bound nanosized vesicles of biologic origin, are known to contain various molecules, e.g., proteins, lipids, and nucleic acids, which contribute to the exosomes' ability to mediate cell-to-cell communication. Recent impediments of artificial nanoparticles in drug delivery, including low cellular uptake, activation of the immune system, and tissue obstacles, have led scientists to engineer exosomes as drug delivery vehicles. Though exosomes possess inherent properties of stability, biocompatibility, low immunogenicity, and capability to cross biological barriers, there is a need to develop technologies that allow the efficient loading of therapeutic materials into exosomes. Here, we introduced a simple peptide-equipped technology that can enhance the cargo-loading potential of exosomes in a mild loading environment. Specifically, a known cell-penetrating peptide, YARA, derived from human immunodeficiency virus-1 trans-activator of transcription, was covalently conjugated with miR-21-5p, a mammalian microRNA. The conjugate YARA-miR-21-5p was then incubated with exosomes, isolated from either mesenchymal stem cells or cancer cells, for loading. Exosomal loading of YARA-miR-21-5p was time-dependent and demonstrated an impressive 18.6-fold increase in efficiency over exosomal loading of miR-21-5p through incubation. After effective cellular uptake, the loaded exosomes rapidly delivered YARA-miR-21-5p into mammalian cells. Relative to unloaded exosomes and free YARA-miR-21-5p, the loaded exosomes significantly enhanced the proliferation, migration, and invasion of human and mouse fibroblasts, which are vital steps in wound healing. This study lays the groundwork for using cell-penetrating peptides as an innovative approach to efficiently load therapeutic cargos, e.g., microRNAs, into exosomes, which can then be employed to deliver the cargos into cells to yield biological effects.

Oral administration of VDAC1-derived small molecule peptides increases circulating testosterone levels in male rats.

Cholesterol is the precursor of all steroid hormones, and the entry of cholesterol into the mitochondria is the rate-limiting step of steroidogenesis. Voltage-dependent anion channel (VDAC1) is an outer mitochondrial protein part of a multiprotein complex that imports cholesterol. We previously reported that intratesticular administration of a 25 amino acid peptide blocking the interaction between 14-3-3ϵ with VDAC1 increased circulating levels of testosterone. This fusion peptide was composed of a HIV-1 transactivator of transcription (TAT) protein transduction domain cell-penetrating peptide, a glycine linker, and amino acids 159-172 of VDAC1 (TV159-172). Here, we describe the development of a family of small molecules that increase circulating testosterone levels after an oral administration. We first characterized an animal model where TV159-172 was delivered subcutaneously. This subcutaneous model allowed us to study the interactions between TV159-172 and the hypothalamus-pituitary-gonadal axis (HPG) and identify the biologically active core of TV159-172. The core consisted of the tetrapeptide RVTQ, which we used as a platform to design synthetic peptide derivatives that can be administered orally. We developed a second animal model to test various derivatives of RVTQ and found 11 active compounds. Dose-response experiments identified 4 synthetic peptides that robustly increased androgen levels in a specific manner. We selected RdVTQ as the leading VDAC1-core derivative and profiled the response across the lifespan of Brown-Norway rats. In summary, we present the development of a new class of therapeutics that act within the HPG axis to increase testosterone levels specifically. This new class of small molecules self-regulates, preventing abuse.

Overcoming Cytosolic Delivery Barriers of Proteins Using Denatured Protein-Conjugated Mesoporous Silica Nanoparticles.

Intracellular delivery of therapeutic proteins has increased advantages over current small-molecule drugs and gene therapies, especially in therapeutic efficacies for a broad spectrum of diseases. Hence, developing the protein therapeutics approach provides a needed alternative. Here, we designed a mesoporous silica nanoparticle (MSN)-mediated protein delivery approach and demonstrated effective intracellular delivery of the denatured superoxide dismutase (SOD) protein, overcoming the delivery challenges and achieving higher enzymatic activity than native SOD-conjugated MSNs. The denatured SOD-conjugated MSN delivery strategy provides benefits of reduced size and steric hindrance, increased protein flexibility without distorting its secondary structure, exposure of the cell-penetrating peptide transactivator of transcription for enhanced efficient delivery, and a change in the corona protein composition, enabling cytosolic delivery. After delivery, SOD displayed a specific activity around threefold higher than in our previous reports. Furthermore, the in vivo biosafety and therapeutic potential for neuron therapy were evaluated, demonstrating the biocompatibility and the effective antioxidant effect in Neuro-2a cells that protected neurite outgrowth from paraquat-induced reactive oxygen species attack. This study offers an opportunity to realize the druggable possibility of cytosolic proteins using MSNs.

Exploring the signal-dependent transcriptional regulation involved in the liver pathology of type 2 diabetes.

Excess glucagon activity in diabetes increases hepatic glucose production via gluconeogenic gene induction, thus exacerbating hyperglycemia. Glucagon receptor-activated cAMP-dependent protein kinase A (PKA) induces proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) expression via the cAMP response element-binding protein (CREB)-regulated transcription coactivator 2 (CRTC2) pathway. Transcriptional coactivator PGC-1α subsequently coactivates transcription factors, such as forkhead box O1 (FoxO1) and hepatocyte nuclear factor 4 alpha (HNF4α), to induce gluconeogenic genes. The current review first summarizes the mechanism by which transcriptional cofactor CBP and p300-activated transactivator with glutamic acid and aspartic acid-rich COOH-terminal domain 2 (CITED2) activates gluconeogenesis via the regulation of PGC-1α and general control of amino acid synthesis protein 5-like 2 (GCN5). Type 2 diabetes is closely linked with non-alcoholic fatty liver disease (NAFLD). Between 10 and 20% of NAFLD progresses to non-alcoholic steatohepatitis (NASH), which can cause liver cirrhosis and can also lead to hepatocellular carcinoma. Liver macrophages are considered to be related to inflammation and fibrosis observed in NASH. This review outlines liver-derived signals underlying the differentiation of liver macrophages and the mechanism of myeloid cell diversification in NASH.

Novel Allosteric Modulator Southern Research Institute-32743 Reverses HIV-1 Transactivator of Transcription-Induced Increase in Dopamine Release in the Caudate Putamen of Inducible Transactivator of Transcription Transgenic Mice.

Development of neurocognitive disorder in human immunodeficiency virus (HIV)-infected patients has been linked to dysregulation of dopamine by the HIV-1 transactivator of transcription (Tat) protein, a negative allosteric modulator of dopamine transporter (DAT). Using fast scan cyclic voltammetry, the present study determined the effects of in vivo Tat expression on dopamine release in the caudate putamen of inducible Tat transgenic (iTat-tg) mice and the impact of a novel DAT allosteric modulator, Southern Research Institute (SRI)-32743, on the Tat effect. We found that 7- or 14-day doxycycline (Dox)-induced Tat expression in iTat-tg mice resulted in a 2-fold increase in phasic but not tonic stimulated baseline dopamine release relative to saline control mice. To determine whether the Tat-induced increase in dopamine release is mediated by DAT regulation, we examined the effect of an in vitro applied DAT inhibitor, nomifensine, on the dopamine release. Nomifensine (1 nM-10 µM) concentration-dependently enhanced phasic stimulated dopamine release in both saline- and Dox-treated iTat-tg mice, while the magnitude of the nomifensine-mediated dopamine release was unchanged between saline and Dox treatment groups. A single systemic administration of SRI-32743 prior to animal sacrifice reversed the increased dopamine release in the baseline of phasic dopamine release and nomifensine-augmented dopamine levels in Dox-treated iTat-tg mice, while SRI-32743 alone did not alter baseline of dopamine release. These findings suggest that Tat expression induced an increase in extracellular dopamine levels by not only inhibiting DAT-mediated dopamine transport but also stimulating synaptic dopamine release. Thus, DAT allosteric modulators may serve as a potential therapeutic intervention for HIV infection-dysregulated dopamine system observed in HIV-1 positive individuals. SIGNIFICANCE STATEMENT: HIV infection-induced dysregulation of the dopaminergic system has been implicated in the development of neurocognitive impairments observed in HIV positive patients. Understanding the mechanisms underlying HIV-1 Tat protein-induced alteration of extracellular dopamine levels will provide insights into the development of molecules that can attenuate Tat interaction with targets in the dopaminergic system. Here, we determined whether Tat alters dopamine release and how the novel DAT allosteric modulator, SRI-32743, impacts dopamine neurotransmission to attenuate Tat-induced effects on extracellular dopamine dynamics.

CHIP ameliorates neuronal damage in H2O2-induced oxidative stress in HT22 cells and gerbil ischemia

Carboxyl terminus of Hsc70-interacting protein (CHIP) is highly conserved and is linked to the connection between molecular chaperones and proteasomes to degrade chaperone-bound proteins. In this study, we synthesized the transactivator of transcription (Tat)-CHIP fusion protein for effective delivery into the brain and examined the effects of CHIP against oxidative stress in HT22 cells induced by hydrogen peroxide (H2O2) treatment and ischemic damage in gerbils by 5 min of occlusion of both common carotid arteries, to elucidate the possibility of using Tat-CHIP as a therapeutic agent against ischemic damage. Tat-CHIP was effectively delivered to HT22 hippocampal cells in a concentration- and time-dependent manner, and protein degradation was confirmed in HT22 cells. In addition, Tat-CHIP significantly ameliorated the oxidative damage induced by 200 μM H2O2 and decreased DNA fragmentation and reactive oxygen species formation. In addition, Tat-CHIP showed neuroprotective effects against ischemic damage in a dose-dependent manner and significant ameliorative effects against ischemia-induced glial activation, oxidative stress (hydroperoxide and malondialdehyde), pro-inflammatory cytokines (interleukin-1β, interleukin-6, and tumor necrosis factor-α) release, and glutathione and its redox enzymes (glutathione peroxidase and glutathione reductase) in the hippocampus. These results suggest that Tat-CHIP could be a therapeutic agent that can protect neurons from ischemic damage.

The Epigenetic Role of miR-124 in HIV-1 Tat- and Cocaine-Mediated Microglial Activation.

HIV-1 and drug abuse have been indissolubly allied as entwined epidemics. It is well-known that drug abuse can hasten the progression of HIV-1 and its consequences, especially in the brain, causing neuroinflammation. This study reports the combined effects of HIV-1 Transactivator of Transcription (Tat) protein and cocaine on miR-124 promoter DNA methylation and its role in microglial activation and neuroinflammation. The exposure of mouse primary microglial cells to HIV-1 Tat (25 ng/mL) and/or cocaine (10 μM) resulted in the significantly decreased expression of primary (pri)-miR-124-1, pri-miR-124-2, and mature miR-124 with a concomitant upregulation in DNMT1 expression as well as global DNA methylation. Our bisulfite-converted genomic DNA sequencing also revealed significant promoter DNA methylation in the pri-miR-124-1 and pri-miR-124-2 in HIV-1 Tat- and cocaine-exposed mouse primary microglial cells. We also found the increased expression of proinflammatory cytokines such as IL1β, IL6 and TNF in the mouse primary microglia exposed to HIV-1 Tat and cocaine correlated with microglial activation. Overall, our findings demonstrate that the exposure of mouse primary microglia to both HIV-1 Tat and cocaine could result in intensified microglial activation via the promoter DNA hypermethylation of miR-124, leading to the exacerbated release of proinflammatory cytokines, ultimately culminating in neuroinflammation.

Delivery of Active AKT1 to Human Cells

Protein kinase B (AKT1) is a serine/threonine kinase and central transducer of cell survival pathways. Typical approaches to study AKT1 biology in cells rely on growth factor or insulin stimulation that activates AKT1 via phosphorylation at two key regulatory sites (Thr308, Ser473), yet cell stimulation also activates many other kinases. To produce cells with specific AKT1 activity, we developed a novel system to deliver active AKT1 to human cells. We recently established a method to produce AKT1 phospho-variants from Escherichia coli with programmed phosphorylation. Here, we fused AKT1 with an N-terminal cell penetrating peptide tag derived from the human immunodeficiency virus trans-activator of transcription (TAT) protein. The TAT-tag did not alter AKT1 kinase activity and was necessary and sufficient to rapidly deliver AKT1 protein variants that persisted in human cells for 24 h without the need to use transfection reagents. TAT-pAKT1T308 induced selective phosphorylation of the known AKT1 substrate GSK-3α, but not GSK-3β, and downstream stimulation of the AKT1 pathway as evidenced by phosphorylation of ribosomal protein S6 at Ser240/244. The data demonstrate efficient delivery of AKT1 with programmed phosphorylation to human cells, thus establishing a cell-based model system to investigate signaling that is dependent on AKT1 activity.

Dynamics and consequences of the HTLV-1 proviral plus-strand burst.

Expression of the transcriptional transactivator protein Tax, encoded on the proviral plus-strand of human T-cell leukaemia virus type 1 (HTLV-1), is crucial for the replication of the virus, but Tax-expressing cells are rarely detected in fresh blood ex vivo. The dynamics and consequences of the proviral plus-strand transcriptional burst remain insufficiently characterised. We combined time-lapse live-cell imaging, single-cell tracking and mathematical modelling to study the dynamics of Tax expression at single-cell resolution in two naturally-infected, non-malignant T-cell clones transduced with a short-lived enhanced green fluorescent protein (d2EGFP) Tax reporter system. Five different patterns of Tax expression were observed during the 30-hour observation period; the distribution of these patterns differed between the two clones. The mean duration of Tax expression in the two clones was 94 and 417 hours respectively, estimated from mathematical modelling of the experimental data. Tax expression was associated with a transient slowing in cell-cycle progression and proliferation, increased apoptosis, and enhanced activation of the DNA damage response pathways. Longer-term follow-up (14 days) revealed an increase in the proportion of proliferating cells and a decrease in the fraction of apoptotic cells as the cells ceased Tax expression, resulting in a greater net expansion of the initially Tax-positive population. Time-lapse live-cell imaging showed enhanced cell-to-cell adhesion among Tax-expressing cells, and decreased cell motility of Tax-expressing cells at the single-cell level. The results demonstrate the within-clone and between-clone heterogeneity in the dynamics and patterns of HTLV-1 plus-strand transcriptional bursts and the balance of positive and negative consequences of the burst for the host cell.

Lipid nanocapsules for the nose-to-brain delivery of the anti-inflammatory bioactive lipid PGD2-G

Here, prostaglandin D2-glycerol ester (PGD2-G) was selected to target neuroinflammation. As PGD2-G is reported to have a short plasmatic half-life, we propose to use lipid nanocapsules (LNC) as vehicle to safely transport PGD2-G to the central nervous system (CNS). PGD2-G-loaded LNC (PGD2-G-LNC) reduced pro-inflammatory cytokine expression in activated microglial cells, even so after crossing a primary olfactory cell monolayer. A single nasal administration of PGD2-G-LNC in lipopolysaccharide (LPS)-treated mice reduced pro-inflammatory cytokine expression in the olfactory bulb. Coating LNC's surface with a cell-penetrating peptide, transactivator of transcription (TAT), increased its accumulation in the brain. Although TAT-coated PGD2-G-LNC modestly exerted its anti-inflammatory effect in a mouse model of multiple sclerosis similar to free PGD2-G after nasal administration, TAT-coated LNC surprisingly reduced the expression of pro-inflammatory chemokines in the CNS. These data propose LNC as an interesting drug delivery tool and TAT-coated PGD2-G-LNC remains a good candidate, in need of further work.

Structural Insights into the Mechanism of HIV-1 Tat Secretion from the Plasma Membrane

Human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription (Tat) is a small, intrinsically disordered basic protein that plays diverse roles in the HIV-1 replication cycle, including promotion of efficient viral RNA transcription. Tat is released by infected cells and subsequently absorbed by healthy cells, thereby contributing to HIV-1 pathogenesis including HIV-associated neurocognitive disorder. It has been shown that, in HIV-1-infected primary CD4 T-cells, Tat accumulates at the plasma membrane (PM) for secretion, a mechanism mediated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). However, the structural basis for Tat interaction with the PM and thereby secretion is lacking. Herein, we employed NMR and biophysical methods to characterize Tat86 (86 amino acids) interactions with PI(4,5)P2 and lipid nanodiscs (NDs). Our data revealed that Arg49, Lys50 and Lys51 (RKK motif) constitute the PI(4,5)P2 binding site, that Tat86 interaction with lipid NDs is dependent on PI(4,5)P2 and phosphatidylserine (PS), and that the arginine-rich motif (RRQRRR) preferentially interacts with PS. Furthermore, we show that Trp11, previously implicated in Tat secretion, penetrates deeply in the membrane; substitution of Trp11 severely reduced Tat86 interaction with membranes. Deletion of the entire highly basic region and Trp11 completely abolished Tat86 binding to lipid NDs. Our data support a mechanism by which HIV-1 Tat secretion from the PM is mediated by a tripartite signal consisting of binding of the RKK motif to PI(4,5)P2, arginine-rich motif to PS, and penetration of Trp11 in the membrane. Altogether, these findings provide new insights into the molecular requirements for Tat binding to membranes during secretion.

Vascular Endothelial Growth Factor Receptor 2: Molecular Mechanism and Therapeutic Potential in Preeclampsia Comorbidity with Human Immunodeficiency Virus and Severe Acute Respiratory Syndrome Coronavirus 2 Infections.

This review explored the role of vascular endothelial growth factor receptor-2 (VEGFR-2) in the synergy of preeclampsia (PE), human immunodeficiency virus (HIV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Downregulation of VEGFR-2 in PE promotes endothelial dysfunction and prevents endothelial cell (EC) migration, proliferation, and differentiation. The HIV-1 accessory protein, tat (trans-activator of transcription), prevents VEGFR-2 signaling via the vascular endothelial growth factor A (VEGF-A) ligand. Combined antiretroviral therapy (cART) may cause immune reconstitution, impaired decidualization, and endothelial injury, thus may be a risk factor for PE development. The VEGF/VEGFR-2 interaction may be associated with SARS-CoV-2-related pulmonary oedema. Endothelial dysfunction and heightened inflammation are both associated with PE, HIV, and SARS-CoV-2 infection; therefore, it is plausible that both characteristics may be exacerbated in the synergy of these events. In addition, this review explored microRNAs (miR) regulating VEGFR-2. An overexpression of miR-126 is evident in PE, HIV, and SARS-CoV-2 infection; thus, modulating the expression of miR-126 may be a therapeutic strategy. However, the involvement of microRNAs in PE, HIV, and SARS-CoV-2 infection needs further investigating. Since these conditions have been evaluated independently, this review attempts to predict their clinical manifestations in their synergy, as well as independently; thereby providing a platform for early diagnosis and therapeutic potential in PE, HIV, and SARS-CoV-2 infection.

Abstract 14364: Protein Kinase C Beta II Inhibitor Provides Cardiovascular Protection When Combined With Conjugated Myristic Acid and Trans-Activator of Transcription in Porcine Ischemia-Reperfusion Injury

Prompt reperfusion is vital to resuscitating ischemic myocardium. However, cardiomyocyte death still occurs due to ischemia-reperfusion (I/R) injury, which is mediated in part by oxidative stress. Major sources of reactive oxygen species (ROS) during I/R are NADPH oxidase (NOX-2) and mitochondria, which are principally activated by protein kinase C βII (PKCβII). Previously, myristic acid (Myr) and trans-activator of transcription (Tat) conjugated PKCβII inhibitor (Myr-Tat-PKCβII-; N-Myr-Tat-CC-SLNPEWNET) exhibited cardioprotective effects in ex vivo rat hearts. In this study, we hypothesize that Myr-Tat-PKCβII- will mitigate cardiac injury in an in vivo porcine myocardial I/R model compared to scrambled peptide controls. Male Yorkshire pigs (38-50kg) underwent regional I(1hr)/R(3hrs) through balloon-assisted occlusion of the second diagonal branch of the left anterior descending coronary artery (LAD) supplying ~40% of the anterior portion of the myocardium. During reperfusion (balloon deflation), a bolus of Myr-Tat-PKCβII- or scrambled control was infused into the LAD. Cardiac function was evaluated as the relative change in ejection fraction (EF) at the end of 3hr reperfusion compared to baseline. Serial measurements of serum creatine phosphokinase (CPK), troponin I, and myoglobin were evaluated to assess cardiac injury. Post-reperfused hearts were stained with Evans Blue dye to identify the area at risk (AR) and 1% triphenyltetrazolium chloride to demarcate the area of necrosis (AN). Infarct size (AN/AR), EF, and cardiac injury markers were analyzed via Student’s t-test. Myr-Tat-PKCβII- preserved EF with a relative change of 1.2±2.8% compared to 8.9±2.2% in control hearts (p<0.05) from mean baseline EF (61.4±0.5%). Myr-Tat-PKCβII- significantly decreased myoglobin levels at 1hr reperfusion (135±132ng/mL, n=4) compared to scrambled control (1022±346ng/mL, n=3 p<0.05). Myr-Tat-PKCβII- reduced infarct size to 13.5±3.9% (n=4) compared to scrambled control hearts (27.5±7.9%, n=6). CPK and troponin I levels were comparable in both groups. These results suggest Myr-Tat-PKCβII- mitigates cardiac injury when given at reperfusion onset. Future studies will examine Myr-Tat-PKCβII- in an 8 week in-vivo, porcine MI survival study.

The HIV-1 Integrase C-Terminal Domain Induces TAR RNA Structural Changes Promoting Tat Binding.

Recent evidence indicates that the HIV-1 Integrase (IN) binds the viral genomic RNA (gRNA), playing a critical role in the morphogenesis of the viral particle and in the stability of the gRNA once in the host cell. By combining biophysical, molecular biology, and biochemical approaches, we found that the 18-residues flexible C-terminal tail of IN acts as a sensor of the peculiar apical structure of the trans-activation response element RNA (TAR), interacting with its hexaloop. We show that the binding of the whole IN C-terminal domain modifies TAR structure, exposing critical nucleotides. These modifications favour the subsequent binding of the HIV transcriptional trans-activator Tat to TAR, finally displacing IN from TAR. Based on these results, we propose that IN assists the binding of Tat to TAR RNA. This working model provides a mechanistic sketch accounting for the emerging role of IN in the early stages of proviral transcription and could help in the design of anti-HIV-1 therapeutics against this new target of the viral infectious cycle.

Bioengineering of green-synthesized TAT peptide-functionalized silver nanoparticles for apoptotic cell-death mediated therapy of breast adenocarcinoma

Green synthesized silver-nanomaterial has exhibited significant advantages as cancer therapeutics due to their exclusive characteristics that cause inducing various forms of cell death; particularly, they have attracted much attention in the treatment of breast cancer. More specifically, the exploration of silver nanoparticles (SNPs) in investigation of cancer treatment is of booming interest due to the opportunity to customize the physicochemical properties of NPs, to enhance their biocompatibility and interactive potentials through surface functionalization. In the present study, first SNPs were fabricated from Staphylococcus aureus cell-free extract, and then the surface modification was executed by conjugating trans-activator of transcription (TAT) peptide, which is a cell-penetrating peptide to enhance the induction of apoptosis in the breast adenocarcinoma cells. The physicochemical characteristics of both virgin SNPs and TAT peptide conjugated SNPs (cSNPs) were evaluated using UV–vis spectrophotometer, FTIR, SEM-EDX, TGA, DSC, and TEM. FTIR analysis revealed the involvement of amide and amines in SNPs synthesis. The SEM analysis confirmed the spherical shape of SNPs, while EDX showed the elemental nature of silver. TGA determined the protein loss at second weight loss of nearly 45%, and DSC confirmed the endothermic phase transition of both SNPs and cSNPs. Moreover, TEM revealed the particle size of about 26.94 nm and 21.94 nm for SNPs and cSNPs, respectively. In regard to the cancer therapeutic potentials of NPs, the cSNPs exhibited potential cytotoxic activity of IC50 (30.05 μg/mL) against MDA-MB-231 breast cancer cells compared to SNPs that induced apoptosis by enhancing the upregulation of p53 protein expression. Internment flow cytometry data confirmed the productions of ROS radicals in the treated cells of the MDA-MB-231 cells. Overall, our study shows that the surface functionalization of SNPs by employing potential inhouse human-derived peptides could be a new and promising approach in cancer therapy.