Transcriptional Transactivation Research Articles

Features of Tat Protein in HIV-1 Sub-Subtype A6 Variants Circulating in the Moscow Region, Russia.

Tat, the trans-activator of transcription, is a multifunctional HIV-1 protein that can induce chronic inflammation and the development of somatic diseases in HIV-infected patients. Natural polymorphisms in Tat can impact the propagation of the inflammatory signal. Currently, Tat is considered an object for creating new therapeutic agents. Therefore, the identification of Tat protein features in various HIV-1 variants is a relevant task. The purpose of the study was to characterize the genetic variations of Tat-A6 in virus variants circulating in the Moscow Region. The authors analyzed 252 clinical samples from people living with HIV (PLWH) with different stages of HIV infection. Nested PCR for two fragments (tat1, tat2) with subsequent sequencing, subtyping, and statistical analysis was conducted. The authors received 252 sequences for tat1 and 189 for tat2. HIV-1 sub-subtype A6 was identified in 250 samples. The received results indicated the features of Tat1-A6 in variants of viruses circulating in the Moscow Region. In PLWH with different stages of HIV infection, C31S in Tat1-A6 was detected with different occurrence rates. It was demonstrated that Tat2-A6, instead of a functional significant 78RGD80 motif, had a 78QRD80 motif. Herewith, G79R in Tat2-A6 was defined as characteristic amino acid substitution for sub-subtype A6. Tat2-A6 in variants of viruses circulating in the Moscow Region demonstrated high conservatism.

Disruption of blood-brain barrier: effects of HIV Tat on brain microvascular endothelial cells and tight junction proteins.

Although the widespread use of antiretroviral therapy (ART) has prolonged the life span of people living with HIV (PLWH), the incidence of HIV-associated neurocognitive disorders (HAND) in PLWH is also gradually increasing, seriously affecting the quality of life for PLWH. However, the pathogenesis of HAND has not been elucidated, which leaves HAND without effective treatment. HIV protein transactivator of transcription (Tat), as an important regulatory protein, is crucial in the pathogenesis of HAND, and its mechanism of HAND has received widespread attention. The blood-brain barrier (BBB) and its cellular component brain microvascular endothelial cells (BMVECs) play a necessary role in protecting the central nervous system (CNS), and their damage associated with Tat is a potential therapeutic target of HAND. In this review, we will study the Tat-mediated damage mechanism of the BBB andpresent multiple lines of evidence related to BMVEC damage caused by Tat.

Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for Sensitive Analysis of Residual Protein Tat Bh1-101 in Lentiviral Vectors for Gene Therapy.

Lentiviral (LV) vector-based gene therapy is gaining popularity for treating a wide range of diseases. Various LV vectors are being developed for transducing cells in cellular gene therapy at St. Jude. Some LV vectors are produced using stable 293T packaging cell lines, which includes gag-pol-rev-tat and virus-glycoprotein. Transactivating factor (transactivator of transcription [Tat]) is a regulatory protein that drastically increases the efficiency of lentiviral transcription. Residual analysis of Tat is critical for gene vector quality and safety. In this work, we developed a highly sensitive liquid chromatography-tandem mass spectrometry method for analysis of residual Tat in Lentivirus as an alternative to enzyme-linked immunosorbent assay. Residual Tat in LV can be accurately quantified with high specificity with a limit of detection of 0.3 ng/mL.

Reducing the Incidence of Post – Myocardial Infarction Heart Failure in an Era of Improved Primary Percutaneous Coronary Intervention through Novel Therapeutics (Protein Kinase C Beta II Inhibitor)

Heart failure (HF) complicating myocardial infarction (MI) remains a major source of morbidity, mortality and a huge health-care burden in the world. The incidence of symptomatic HF after primary percutaneous coronary intervention (PPCI) has been widely reported. PPCI has improved early survival of MI, but its impact on downstream HF is deliberated. Infarct size is the major determinant of the development of HF and prognosis. Efforts to reduce the size will therefore have a significant impact on the incidence of HF after PPCI. Resuscitation of the ischemic myocardium, heavily rests on timely reperfusion. However, cardiomyocyte death still occurs due to ischemic/reperfusion (I/R) injury mediated by oxidative stress. This phenomenon is known to largely contribute to the size of infarct. Activation by protein kinase C-βII (PKCβII) is attributed to the generation of reactive oxygen species (ROS) during this process and inhibition of this drive could salvage the myocardial tissue and subsequently preserve cardiac function. We hypothesized that myristic acid (Myr) and trans-activator of transcription (Tat) conjugated PKCβII inhibitor (Myr-Tat-PKCβII-) will exhibit robust cardioprotective effects in an in vivo porcine model, reflective of our ex vivo rat hearts model. Male Yorkshire pigs underwent regional I/R by occluding the second branch of the left anterior descending artery (LAD). This was achieved in the catheterization laboratory guided by fluoroscopy. A bolus of Myr-Tat-PKCβII- or scrambled control was infused into the LAD during reperfusion. Cardiac function was evaluated by an echocardiogram during the entire procedure, and assessed as the relative change at the end of 3hr reperfusion compared to baseline. Post-reperfused hearts were stained with Evans Blue dye to identify the area a risk and 1% triphenyltetrazolium chloride to delineate the area of necrosis. Infarct size and ejection fraction (EF) were analyzed via Student’s t- test. From mean baseline EF (61.4±0.5%), 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) at the end of reperfusion. Myr-Tat-PKCβII- reduced infarct size to 13.5±3.9% (n=4) compared to scrambled control hearts (27.5 ±7.9%, n=6). These results suggest Myr-Tat-PKCβII- significantly preserve EF after PPCI and reduce infarct size when given at reperfusion onset. This study gives a solid premise for future examination of Myr-Tat-PKCβII- in an 8 week in-vivo, porcine MI survival study.

Discontinuous L-binding motifs in the transactivation domain of the vesicular stomatitis virus P protein are required for terminal de novo transcription initiation by the L protein.

The phospho- (P) protein, the co-factor of the RNA polymerase large (L) protein, of vesicular stomatitis virus (VSV, a prototype of nonsegmented negative-strand RNA viruses) plays pivotal roles in transcription and replication. However, the precise mechanism underlying the transcriptional transactivation by the P protein has remained elusive. Here, using an in vitro transcription system and a series of deletion mutants of the P protein, we mapped a region encompassing residues 51-104 as a transactivation domain (TAD) that is critical for terminal de novo initiation, the initial step of synthesis of the leader RNA and anti-genome/genome, with the L protein. Site-directed mutagenesis revealed that conserved amino acid residues in three discontinuous L-binding sites within the TAD are essential for the transactivation activity of the P protein or important for maintaining its full activity. Importantly, relative inhibitory effects of TAD point mutations on synthesis of the full-length leader RNA and mRNAs from the 3'-terminal leader region and internal genes, respectively, of the genome were similar to those on terminal de novo initiation. Furthermore, any of the examined TAD mutations did not alter the gradient pattern of mRNAs synthesized from internal genes, nor did they induce the production of readthrough transcripts. These results suggest that these TAD mutations impact mainly terminal de novo initiation but rarely other steps (e.g., elongation, termination, internal initiation) of single-entry stop-start transcription. Consistently, the mutations of the essential or important amino acid residues within the P TAD were lethal or deleterious to VSV replication in host cells. IMPORTANCE RNA-dependent RNA polymerase L proteins of nonsegmented negative-strand RNA viruses belonging to the Mononegavirales order require their cognate co-factor P proteins or their counterparts for genome transcription and replication. However, exact roles of these co-factor proteins in modulating functions of L proteins during transcription and replication remain unknown. In this study, we revealed that three discrete L-binding motifs within a transactivation domain of the P protein of vesicular stomatitis virus, a prototypic nonsegmented negative-strand RNA virus, are required for terminal de novo initiation mediated by the L protein, which is the first step of synthesis of the leader RNA as well as genome/anti-genome.

HIV-1 subtype B Tat enhances NOTCH3 signaling in astrocytes to mediate oxidative stress, inflammatory response, and neuronal apoptosis.

NOTCH receptors are relevant to multiple neurodegenerative diseases. However, the roles and mechanisms of NOTCH receptors in HIV-associated neurocognitive disorder (HAND) remain largely unclear. Transactivator of transcription (Tat) induces oxidative stress and inflammatory response in astrocytes, thereby leading to neuronal apoptosis in the central nervous system. We determined that NOTCH3 expression was upregulated during subtype B or C Tat expression in HEB astroglial cells. Moreover, bioinformatics analysis of the Gene Expression Omnibus (GEO) dataset revealed that NOTCH3 mRNA expression in the frontal cortex tissues of HIV encephalitis patients was higher than that of HIV control patients. Of note, subtype B Tat, rather than subtype C Tat, interacted with the extracellular domain of the NOTCH3 receptor, thus activating NOTCH3 signaling. Downregulation of NOTCH3 attenuated subtype B Tat-induced oxidative stress and reactive oxygen species generation. In addition, we demonstrated that NOTCH3 signaling facilitated subtype B Tat-activated NF-κB signaling pathway, thereby mediating pro-inflammatory cytokines IL-6 and TNF-α production. Furthermore, downregulation of NOTCH3 in HEB astroglial cells protected SH-SY5Y neuronal cells from astrocyte-mediated subtype B Tat neurotoxicity. Taken together, our study clarifies the potential role of NOTCH3 in subtype B Tat-induced oxidative stress and inflammatory response in astrocytes, which could be a novel therapeutic target for the relief of HAND.

HIV Tat-mediated altered oligodendrocyte maturation involves autophagy-lysosomal dysfunction

Abstract Objectives The current study was undertaken to understand the underlying molecular mechanism(s) by which HIV Transactivator of transcription (Tat) alters oligodendrocyte maturation through the generation of reactive oxygen species (ROS), impairment of lysosomal functioning, and dysregulation of autophagy. Methods We exposed primary rat immature oligodendrocytes to HIV Tat and utilized various experimental techniques to assess its effects on oligodendrocytes maturation. We measured ROS levels, assessed lysosomal membrane potential, determined cathepsin D activity, and analyzed the expression of autophagy-related markers. Furthermore, we investigated the potential of ROS scavengers and lysosomal protectants to mitigate the damaging effects of HIV Tat on oligodendrocytes maturation. Results Exposure of primary rat immature oligodendrocytes to HIV Tat significantly increased ROS levels, indicating the induction of oxidative stress. This oxidative stress impaired lysosomal functioning, as evidenced by a substantial increase in lysosomal membrane potential and a decrease in cathepsin D activity. Compromised lysosomal function resulted in dysregulated autophagy, which was confirmed by increased expression of SQSTM1. However, the administration of ROS scavengers and lysosomal protectants effectively attenuated the detrimental effects of HIV Tat on oligodendrocytes maturation. Conclusions Our findings demonstrate that HIV Tat exposure induces oxidative stress, impairs lysosomal functioning, and dysregulates autophagy in oligodendrocytes. These molecular changes likely contribute to the altered maturation of oligodendrocytes observed in HIV-infected individuals. Understanding these underlying mechanisms provides valuable insights into the pathogenesis of HIV-associated neurocognitive disorders and highlights the potential of therapeutic strategies targeting ROS scavenging and lysosomal protection as adjunctive approaches for managing such complications in HIV +ve individuals.

A Transcriptome Array-Based Approach Links Proteinuria and Distinct Molecular Signatures to Intrarenal Expression of Type I Interferon IFNA5 in Lupus Nephritis

In systemic lupus erythematosus (SLE), the relevance of non-hematopoietic sources of type I interferon in human autoimmunity has recently been recognized. Particularly, type I interferon production precedes autoimmunity in early skin lesions related to SLE. However, the relevance of intrarenal type I interferon expression has not been shown in lupus nephritis. From transcriptome array datasets, median-centered log2 mRNA expression levels of IFNα (IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21), IFNω (IFNW1), and IFNβ (IFNB1) in lupus nephritis were extracted specifically from microdissected tubulointerstitial (n = 32) and glomerular compartments (n = 32). We found an association between proteinuria and tubulointerstitial expression of type I interferon IFNA5 (p = 0.0142), while all others were not significantly associated. By contrast, no such correlation was observed between proteinuria and any type I interferon expression in the glomerular compartment in lupus nephritis. Interestingly, there was no difference between female and male patients (p = 0.8237) and no association between type I interferon IFNA5 expression and kidney function or lupus nephritis progression. Finally, we identified distinct molecular signatures involved in transcriptional regulation (GLI protein-regulated transcription, IRF7 activation, and HSF1-dependent transactivation) and receptor signaling (BMP signaling and GPCR ligand binding) in association with tubulointerstitial expression of type I interferon IFNA5 in the kidney. In summary, this transcriptome array-based approach links proteinuria to the tubulointerstitial expression of type I interferon IFNA5 in lupus nephritis. Because type I interferon receptor subunit I antagonism has recently been investigated in active SLE, the current study further emphasizes the role of type I interferons in lupus nephritis and might also be of relevance for mechanistic studies.

Genetic variation of the HIV-1 subtype C transmitted/founder viruses long terminal repeat elements and the impact on transcription activation potential and clinical disease outcomes.

A genetic bottleneck is a hallmark of HIV-1 transmission such that only very few viral strains, termed transmitted/founder (T/F) variants establish infection in a newly infected host. Phenotypic characteristics of these variants may determine the subsequent course of disease. The HIV-1 5' long terminal repeat (LTR) promoter drives viral gene transcription and is genetically identical to the 3' LTR. We hypothesized that HIV-1 subtype C (HIV-1C) T/F virus LTR genetic variation is a determinant of transcriptional activation potential and clinical disease outcome. The 3'LTR was amplified from plasma samples of 41 study participants acutely infected with HIV-1C (Fiebig stages I and V/VI). Paired longitudinal samples were also available at one year post-infection for 31 of the 41 participants. 3' LTR amplicons were cloned into a pGL3-basic luciferase expression vector, and transfected alone or together with Transactivator of transcription (tat) into Jurkat cells in the absence or presence of cell activators (TNF-α, PMA, Prostratin and SAHA). Inter-patient T/F LTR sequence diversity was 5.7% (Renge: 2-12) with subsequent intrahost viral evolution observed in 48.4% of the participants analyzed at 12 months post-infection. T/F LTR variants exhibited differential basal transcriptional activity, with significantly higher Tat-mediated transcriptional activity compared to basal (p<0.001). Basal and Tat-mediated T/F LTR transcriptional activity showed significant positive correlation with contemporaneous viral loads and negative correlation with CD4 T cell counts (p<0.05) during acute infection respectively. Furthermore, Tat-mediated T/F LTR transcriptional activity significanly correlated positively with viral load set point and viral load; and negatively with CD4 T cell counts at one year post infection (all p<0.05). Lastly, PMA, Prostratin, TNF-α and SAHA cell stimulation resulted in enhanced yet heterologous transcriptional activation of different T/F LTR variants. Our data suggest that T/F LTR variants may influence viral transcriptional activity, disease outcomes and sensitivity to cell activation, with potential implications for therapeutic interventions.

Demonstration of the Early Cardiac Bioavailability of a Non-Specific Cell-Targeted Peptide Using Radionuclide-Based Imaging In Vivo.

The cardiac bioavailability of peptide drugs that inhibit harmful intracellular protein-protein interactions in cardiovascular diseases remains a challenging task in drug development. This study investigates whether a non-specific cell-targeted peptide drug is available in a timely manner at its intended biological destination, the heart, using a combined stepwise nuclear molecular imaging approach. An octapeptide (heart8P) was covalently coupled with the trans-activator of transcription (TAT) protein transduction domain residues 48-59 of human immunodeficiency virus-1 (TAT-heart8P) for efficient internalization into mammalian cells. The pharmacokinetics of TAT-heart8P were evaluated in dogs and rats. The cellular internalization of TAT-heart8P-Cy(5.5) was examined on cardiomyocytes. The real-time cardiac delivery of 68Ga-NODAGA-TAT-heart8P was tested in mice under physiological and pathological conditions. Pharmacokinetic studies of TAT-heart8P in dogs and rats revealed a fast blood clearance, high tissue distribution, and high extraction by the liver. TAT-heart-8P-Cy(5.5) was rapidly internalized in mouse and human cardiomyocytes. Correspondingly, organ uptake of hydrophilic 68Ga-NODAGA-TAT-heart8P occurred rapidly after injection with an initial cardiac bioavailability already 10 min post-injection. The saturable cardiac uptake was revailed by the pre-injection of the unlabeled compound. The cardiac uptake of 68Ga-NODAGA-TAT-heart8P did not change in a model of cell membrane toxicity. This study provides a sequential stepwise workflow to evaluate the cardiac delivery of a hydrophilic, non-specific cell-targeting peptide. 68Ga-NODAGA-TAT-heart8P showed rapid accumulation in the target tissue early after injection. The implementation of PET/CT radionuclide-based imaging methodology as a means to assess effective and temporal cardiac uptake represents a useful and critical application in drug development and pharmacological research and can be extended to the evaluation of comparable drug candidates.

Role of Dysregulated Autophagy in HIV Tat, Cocaine, and cART Mediated NLRP3 Activation in Microglia.

Despite the ability of combination antiretroviral therapy (cART) to suppress viremia, there is persistence low levels of HIV proteins such as Transactivator of transcription (Tat) in the central nervous system (CNS), contributing to glial activation and neuroinflammation. Accumulating evidence also implicates the role of drugs of abuse in exacerbating neurological complications associated with HIV-1. The combined effects of HIV Tat, drugs of abuse, and cART can thus create a toxic milieu in the CNS. The present study investigated the combinatorial effects of HIV-Tat, cocaine, and cART on autophagy and NLRP3 inflammasome activation. We selected a combination of three commonly used cART regimens: tenofovir, emtricitabine, and dolutegravir. Our results demonstrated that exposure of mouse primary microglia (MPMs) to these agents-HIV Tat (25ng/ml), cocaine (1μM), and cART (1μM each) resulted in upregulation of autophagy markers: Beclin1, LC3B-II, and SQSTM1 with impaired lysosomal functioning involving increased lysosomal pH, decreased LAMP2 and cathepsin D, ultimately leading to dysregulated autophagy. Our findings also demonstrated activation of the NLRP3 signaling in microglia exposed to these agents. We further demonstrated that gene silencing of key autophagy protein BECN1 significantly blocked NLRP3-mediated activation of microglia. Silencing of NLRP3, however, failed to block HIV Tat, cocaine, and cART-mediated dysregulation of the autophagy-lysosomal axis; these in vitro phenomena were also validated in vivo using iTat mice administered cocaine and cART. This study thus underscores the cooperative effects of HIV Tat, cocaine, and cART in exacerbating microglial activation involving dysregulated autophagy and activation of the NLRP3 inflammasome signaling.

HIV-1 Tat impairs the establishment and maintenance of latency in a quiescent T cell line model of HIV infection

Abstract Human Immunodeficiency Virus (HIV) persists as a transcriptionally silent, latent provirus despite optimal therapy. Latently infected cells avoid immune detection and viral cytopathic effects becoming a long-lived reservoir of HIV. To elucidate factors responsible for the establishment of latency, our lab developed two novel HIV-1 reporter constructs (89.6 VT1 and 454 VT2). Studies using these reporters demonstrated that the multiplicity of infection (MOI) (the ratio of viral particles to cells) strongly correlated with the proportion of active versus latent infection in a “quiescent” T cell line. This observation suggested the hypothesis that at low viral inoculum, the availability of a viral factor, such as the HIV transactivator of transcription (Tat), was limiting and affected the likelihood that active versus latent infection was established. To test this, we (1) examined relative tat levels in reporter viruses that established different proportions of latent versus active infection (2) measured active versus latent infection in a cell line that constitutively overexpressed tat and (3) modified the dual reporter 89.6 VT1 to overexpress tat after an IRES element. In every case, higher tat levels correlated with increased active infection and a reduction in dependency of active infection on the amount of inoculum. Ongoing studies will test the extent to which Tat levels vary in native HIV proviral genomes. These studies provide insight into the molecular mechanisms that govern latency establishment at high versus low viral infection conditions and may shed light on new approaches to eradicate latently infected cells. Supported by grants from NIH/ NIAID (R01 AI148084 S1)

Investigating HIV-1 TAT protein role in Atherogenesis

Abstract With the recent development of antiretroviral therapy, HIV patients can live a long and normal lifespan. Recent evidence demonstrates an increased incidence of developing cardiovascular diseases (CVD) specifically atherosclerosis in aged HIV-infected patients. The adaptive and innate immune responses play a complex specific role in atherosclerotic plaque burden. However, specific mechanisms of the implication of the immune system in HIV-accelerated atherosclerosis are unclear. The protein Trans-Activator of Transcription–TAT is strongly associated with HIV infection as it enhances the efficiency of viral transcription. To understand the contributions of TAT protein in HIV-associated atherogenesis, a doxycycline-inducible astrocyte-specific HIV-1 TAT transgenic mouse was used. An injection of recombinant adeno-associated virus vector (AAV) encoding a gain-of-function mutant PCSK9 into mice and feeding with high fat with added cholesterol (DDC) and doxycycline (Dox) was used to promote atherosclerosis and TAT expression. After 16 wks of feeding, various tissues were collected and used for flow cytometry analysis (FACS) for immune profiling. Our preliminary data suggest that TAT is involved in the regulation of peripheral blood neutrophil numbers and therefore can serve as a regulator of inflammation in HIV-associated atherosclerosis. Future analysis will provide insight into potential mechanisms of HIV TAT-dependent involvement in atherosclerosis development and progression. This work was supported by project –initiation funds from the Center for Integrative Neuroscience and Inflammatory Diseases at EVMS and NIH NHLBI R01HL142129-04S1 grant.

Iron Saturation Drives Lactoferrin Effects on Oxidative Stress and Neurotoxicity Induced by HIV-1 Tat.

The Trans-Activator of Transcription (Tat) of Human Immunodeficiency Virus (HIV-1) is involved in virus replication and infection and can promote oxidative stress in human astroglial cells. In response, host cells activate transcription of antioxidant genes, including a subunit of System Xc- cystine/glutamate antiporter which, in turn, can trigger glutamate-mediated excitotoxicity. Here, we present data on the efficacy of bovine Lactoferrin (bLf), both in its native (Nat-bLf) and iron-saturated (Holo-bLf) forms, in counteracting oxidative stress in U373 human astroglial cells constitutively expressing the viral protein (U373-Tat). Our results show that, dependent on iron saturation, both Nat-bLf and Holo-bLf can boost host antioxidant response by up-regulating System Xc- and the cell iron exporter Ferroportin via the Nuclear factor erythroid 2-related factor (Nrf2) pathway, thus reducing Reactive Oxygen Species (ROS)-mediated lipid peroxidation and DNA damage in astrocytes. In U373-Tat cells, both forms of bLf restore the physiological internalization of Transferrin (Tf) Receptor 1, the molecular gate for Tf-bound iron uptake. The involvement of astrocytic antioxidant response in Tat-mediated neurotoxicity was evaluated in co-cultures of U373-Tat with human neuronal SH-SY5Y cells. The results show that the Holo-bLf exacerbates Tat-induced excitotoxicity on SH-SY5Y, which is directly dependent on System-Xc- upregulation, thus highlighting the mechanistic role of iron in the biological activities of the glycoprotein.

Disruption of the ADAM17/NF-κB feedback loop in astrocytes ameliorates HIV-1 Tat-induced inflammatory response and neuronal death.

A disintegrin and metalloproteinases (ADAMs) are involved in multiple neurodegenerative diseases. However, the roles and mechanisms of ADAMs in HIV-associated neurocognitive disorder (HAND) remain unclear. Transactivator of transcription (Tat) induces inflammatory response in astrocytes, thereby leading to neuronal apoptosis in the central nervous system. In this study, we determined that ADAM17 expression was upregulated during soluble Tat stimulus in HEB astroglial cells. Inhibition of ADAM17 suppressed Tat-induced pro-inflammatory cytokines production and rescued the astrocytes-derived conditioned media (ACM)-mediated SH-SY5Y neural cells apoptosis. Moreover, ADAM17 mediated Tat-triggered inflammatory response in a NF-κB-dependent manner. Conversely, Tat induced ADAM17 expression via NF-κB signaling pathway. In addition, pharmacological inhibition of NF-κB signaling inhibited Tat-induced inflammatory response, which could be rescued by overexpression of ADAM17. Taken together, our study clarifies the potential role of the ADAM17/NF-κB feedback loop in Tat-induced inflammatory response in astrocytes and the ACM-mediated neuronal death, which could be a novel therapeutic target for relief of HAND.

Exosomal IRF1-loaded rat adipose-derived stem cell sheet contributes to wound healing in the diabetic foot ulcers

BackgroundCell-based therapy has been recognized as a novel technique for the management of diabetic foot ulcers, and cell-sheet engineering leads to improved efficacy in cell transplantation. This study aims to explore the possible molecular mechanism of the rat adipose-derived stem cell (ASC) sheet loaded with exosomal interferon regulatory factor 1 (IRF1) in foot wound healing.MethodsRats were rendered diabetic with streptozotocin, followed by measurement of miR-16-5p expression in wound tissues. Relationship between IRF1, microRNA (miR)-16-5p, and trans-acting transcription factor 5 (SP5) was analyzed using luciferase activity, RNA pull-down, and chromatin immunoprecipitation assays. IRF1 was overexpressed in rat ASCs (rASCs) or loaded onto the rASC sheet, and then exosomes were extracted from rASCs. Accordingly, we assessed the effects of IRF1-exosome or IRF1-rASC sheet on the proliferation and migration of the fibroblasts along with endothelial cell angiogenesis.ResultsmiR-16-5p was poorly expressed in the wound tissues of diabetic rats. Overexpression of miR-16-5p promoted fibroblast proliferation and migration as well as endothelial cell angiogenesis, thus expediting wound healing. IRF1 was an upstream transcription factor that could bind to the miR-16-5p promoter and increase its expression. In addition, SP5 was a downstream target gene of miR-16-5p. IRF1-exosome from rASCs or the IRF1-rASC sheet facilitated the foot wound healing in diabetic rats through miR-16-5p-dependent inhibition of SP5.ConclusionThe present study demonstrates that exosomal IRF1-loaded rASC sheet regulates miR-16-5p/SP5 axis to facilitate wound healing in diabetic rats, which aids in development of stem cell-based therapeutic strategies for diabetic foot wounds.

Drosophila DAxud1 Has a Repressive Transcription Activity on Hsp70 and Other Heat Shock Genes.

Drosophila melanogaster DAxud1 is a transcription factor that belongs to the Cysteine Serine Rich Nuclear Protein (CSRNP) family, conserved in metazoans, with a transcriptional transactivation activity. According to previous studies, this protein promotes apoptosis and Wnt signaling-mediated neural crest differentiation in vertebrates. However, no analysis has been conducted to determine what other genes it might control, especially in connection with cell survival and apoptosis. To partly answer this question, this work analyzes the role of Drosophila DAxud1 using Targeted-DamID-seq (TaDa-seq), which allows whole genome screening to determine in which regions it is most frequently found. This analysis confirmed the presence of DAxud1 in groups of pro-apoptotic and Wnt pathway genes, as previously described; furthermore, stress resistance genes that coding heat shock protein (HSP) family genes were found as hsp70, hsp67, and hsp26. The enrichment of DAxud1 also identified a DNA-binding motif (AYATACATAYATA) that is frequently found in the promoters of these genes. Surprisingly, the following analyses demonstrated that DAxud1 exerts a repressive role on these genes, which are necessary for cell survival. This is coupled with the pro-apoptotic and cell cycle arrest roles of DAxud1, in which repression of hsp70 complements the maintenance of tissue homeostasis through cell survival modulation.

A Cdk5-derived peptide inhibits Cdk5/p25 activity and improves neurodegenerative phenotypes

Aberrant activity of cyclin-dependent kinase (Cdk5) has been implicated in various neurodegenerative diseases. This deleterious effect is mediated by pathological cleavage of the Cdk5 activator p35 into the truncated product p25, leading to prolonged Cdk5 activation and altered substrate specificity. Elevated p25 levels have been reported in humans and rodents with neurodegeneration, and the benefit of genetically blocking p25 production has been demonstrated previously in rodent and human neurodegenerative models. Here, we report a 12-amino-acid-long peptide fragment derived from Cdk5 (Cdk5i) that is considerably smaller than existing peptide inhibitors of Cdk5 (P5 and CIP) but shows high binding affinity toward the Cdk5/p25 complex, disrupts the interaction of Cdk5 with p25, and lowers Cdk5/p25 kinase activity. When tagged with a fluorophore (FITC) and the cell-penetrating transactivator of transcription (TAT) sequence, the Cdk5i-FT peptide exhibits cell- and brain-penetrant properties and confers protection against neurodegenerative phenotypes associated with Cdk5 hyperactivity in cell and mouse models of neurodegeneration, highlighting Cdk5i's therapeutic potential.

Molecular Mechanism of Sirtuin 1 Inhibition by Human Immunodeficiency Virus 1 Tat Protein.

Sirtuins are NAD+-dependent protein lysine deacylases implicated in metabolic regulation and aging-related dysfunctions. The nuclear isoform Sirt1 deacetylates histones and transcription factors and contributes, e.g., to brain and immune cell functions. Upon infection by human immunodeficiency virus 1 (HIV1), Sirt1 deacetylates the viral transactivator of transcription (Tat) protein to promote the expression of the viral genome. Tat, in turn, inhibits Sirt1, leading to the T cell hyperactivation associated with HIV infection. Here, we describe the molecular mechanism of Tat-dependent sirtuin inhibition. Using Tat-derived peptides and recombinant Tat protein, we mapped the inhibitory activity to Tat residues 34-59, comprising Tat core and basic regions and including the Sirt1 deacetylation site Lys50. Tat binds to the sirtuin catalytic core and inhibits Sirt1, Sirt2, and Sirt3 with comparable potencies. Biochemical data and crystal structures of sirtuin complexes with Tat peptides reveal that Tat exploits its intrinsically extended basic region for binding to the sirtuin substrate binding cleft through substrate-like β-strand interactions, supported by charge complementarity. Tat Lys50 is positioned in the sirtuin substrate lysine pocket, although binding and inhibition do not require prior acetylation and rely on subtle differences to the binding of regular substrates. Our results provide mechanistic insights into sirtuin regulation by Tat, improving our understanding of physiological sirtuin regulation and the role of this interaction during HIV1 infection.

CD3+CD56+ and CD3-CD56+ lymphocytes in the cerebrospinal fluid of persons with HIV-1 subtypes B and C.

The transactivator of transcription (Tat) is a HIV regulatory protein which promotes viral replication and chemotaxis. HIV-1 shows extensive genetic diversity, HIV-1 subtype C being the most dominant subtype in the world. Our hypothesis is the frequency of CSF CD3+CD56+ and CD3-CD56dim is reduced in HIV-1C compared to HIV-1B due to the Tat C30S31 substitution in HIV-1C. 34 CSF and paired blood samples (PWH, n=20; PWoH, n=14) were studied. In PWH, the percentage of CD3+CD56+ was higher in CSF than in blood (p<0.001), comparable in both compartments in PWoH (p=0.20). The proportion of CD3-CD56dim in CSF in PWH was higher than PWoH (p=0.008). There was no subtype differences. These results showed CNS compartmentalization of NKT cell response in PWH.