A Step-by-Step Guide for the Production of Recombinant Fluorescent TAT-HA-Tagged Proteins and their Transduction into Mammalian Cells.

Investigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS-PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers. In this protocol, we describe step-by-step the engineering of a recombinant protein with various tags: TAT-HA (trans-activator of transduction-hemagglutinin), 6×His and EGFP (enhanced green fluorescent protein) or mCherry. Fusion proteins are produced in E. coli BL21(DE3) cells and purified by immobilized metal affinity chromatography (IMAC) using a Ni-nitrilotriacetic acid (NTA) column. Then, tagged recombinant proteins are introduced into cultured animal cells by using the penetrating peptide TAT-HA. Here, we present a thorough protocol providing a detailed guide encompassing every critical step from plasmid DNA molecular assembly to protein expression and subsequent purification and outlines the conditions necessary for protein transduction technology into animal cells in a comprehensive manner. We believe that this protocol will be a valuable resource for researchers seeking an exhaustive, step-by-step guide for the successful production and purification of recombinant proteins and their entry by transduction within living cells. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA cloning, molecular assembly strategies, and protein production Basic Protocol 2: Protein purification Basic Protocol 3: Protein transduction in mammalian cells.

Potent latency reversal by Tat RNA-containing nanoparticle enables multi-omic analysis of the HIV-1 reservoir.

The development of latency reversing agents that potently reactivate HIV without inducing global T cell activation would benefit the field of HIV reservoir research and could pave the way to a functional cure. Here, we explore the reactivation capacity of a lipid nanoparticle containing Tat mRNA (Tat-LNP) in CD4 T cells from people living with HIV undergoing antiretroviral therapy (ART). When combined with panobinostat, Tat-LNP induces latency reversal in a significantly higher proportion of latently infected cells compared to PMA/ionomycin (≈ 4-fold higher). We demonstrate that Tat-LNP does not alter the transcriptome of CD4 T cells, enabling the characterization of latently infected cells in their near-native state. Upon latency reversal, we identify transcriptomic differences between infected cells carrying an inducible provirus and non-infected cells (e.g. LINC02964, GZMA, CCL5). We confirm the transcriptomic differences at the protein level and provide evidence that the long non-coding RNA LINC02964 plays a role in active HIV infection. Furthermore, p24+ cells exhibit heightened PI3K/Akt signaling, along with downregulation of protein translation, suggesting that HIV-infected cells display distinct signatures facilitating their long-term persistence. Tat-LNP represents a valuable research tool for in vitro reservoir studies as it greatly facilitates the in-depth characterization of HIV reservoir cells' transcriptome and proteome profiles.

HIV-1 Tat Induces Dysregulation of PGC1-Alpha and Sirtuin 3 Expression in Neurons: The Role of Mitochondrial Biogenesis in HIV-Associated Neurocognitive Disorder (HAND).

During the antiretroviral era, individuals living with HIV continue to experience milder forms of HIV-associated neurocognitive disorder (HAND). Viral proteins, including Tat, play a pivotal role in the observed alterations within the central nervous system (CNS), with mitochondrial dysfunction emerging as a prominent hallmark. As a result, our objective was to examine the expression of genes associated with mitophagy and mitochondrial biogenesis in the brain exposed to the HIV-1 Tat protein. We achieved this by performing bilateral stereotaxic injections of 100 ng of HIV-1 Tat into the hippocampus of Sprague-Dawley rats, followed by immunoneuromagnetic cell isolation. Subsequently, we assessed the gene expression of Ppargc1a, Pink1, and Sirt1-3 in neurons using RT-qPCR. Additionally, to understand the role of Tert in telomeric dysfunction, we quantified the activity and expression of Tert. Our results revealed that only Ppargc1a, Pink1, and mitochondrial Sirt3 were downregulated in response to the presence of HIV-1 Tat in hippocampal neurons. Interestingly, we observed a reduction in the activity of Tert in the experimental group, while mRNA levels remained relatively stable. These findings support the compelling evidence of dysregulation in both mitophagy and mitochondrial biogenesis in neurons exposed to HIV-1 Tat, which in turn induces telomeric dysfunction.

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.

Tat-CIAPIN1 Prevents Pancreatic ß-Cell Death in hIAPP-Induced RINm5F Cells and T2DM Animal Model.

It is well known that the cytokine-induced apoptosis inhibitor 1 (CIAPIN1) protein plays an important role in biological progresses as an anti-apoptotic protein. Human islet amyloid peptide (hIAPP), known as amylin, is caused to pancreatic ß-cell death in type 2 diabetes mellitus (T2DM). However, the function of CIAPIN1 protein on T2DM is not yet well studied. Therefore, we investigated the effects of CIAPIN1 protein on a hIAPP-induced RINm5F cell and T2DM animal model induced by a high-fat diet (HFD) and streptozotocin (STZ). The Tat-CIAPIN1 protein reduced the activation of mitogen-activated protein kinase (MAPK) and regulated the apoptosis-related protein expression levels including COX-2, iNOS, Bcl-2, Bax, and Caspase-3 in hIAPP-induced RINm5F cells. In a T2DM mice model, the Tat-CIAPIN1 protein ameliorated the pathological changes of pancreatic ß-cells and reduced the fasting blood glucose, body weight and hemoglobin Alc (HbAlc) levels. In conclusion, the Tat-CIAPIN1 protein showed protective effects against T2DM by protection of ß-cells via inhibition of hIAPP toxicity and by regulation of a MAPK signal pathway, suggesting CIAPIN1 protein can be a therapeutic protein drug candidate by beneficial regulation of T2DM.

Tat-RAN attenuates brain ischemic injury in hippocampal HT-22 cells and ischemia animal model.

Oxidative stress plays a key role in the pathogenesis of neuronal injury, including ischemia. Ras-related nuclear protein (RAN), a member of the Ras superfamily, involves in a variety of biological roles, such as cell division, proliferation, and signal transduction. Although RAN reveals antioxidant effect, its precise neuroprotective mechanisms are still unclear. Therefore, we investigated the effects of RAN on HT-22 cell which were exposed to H2O2-induced oxidative stress and ischemia animal model by using the cell permeable Tat-RAN fusion protein. We showed that Tat-RAN transduced into HT-22 cells, and markedly inhibited cell death, DNA fragmentation, and reactive oxygen species (ROS) generation under oxidative stress. This fusion protein also controlled cellular signaling pathways, including mitogen-activated protein kinases (MAPKs), NF-κB, and apoptosis (Caspase-3, p53, Bax and Bcl-2). In the cerebral forebrain ischemia animal model, Tat-RAN significantly inhibited both neuronal cell death, and astrocyte and microglia activation. These results indicate that RAN significantly protects against hippocampal neuronal cell death, suggesting Tat-RAN will help to develop the therapies for neuronal brain diseases including ischemic injury.

Protective Effects of a Jellyfish-Derived Thioredoxin Fused with Cell-Penetrating Peptide TAT-PTD on H2O2-Induced Oxidative Damage.

Thioredoxin (Trx) plays a critical role in maintaining redox balance in various cells and exhibits anti-oxidative, anti-apoptotic, and anti-inflammatory effects. However, whether exogenous Trx can inhibit intracellular oxidative damage has not been investigated. In previous study, we have identified a novel Trx from the jellyfish Cyanea capillata, named CcTrx1, and confirmed its antioxidant activities in vitro. Here, we obtained a recombinant protein, PTD-CcTrx1, which is a fusion of CcTrx1 and protein transduction domain (PTD) of HIV TAT protein. The transmembrane ability and antioxidant activities of PTD-CcTrx1, and its protective effects against H2O2-induced oxidative damage in HaCaT cells were also detected. Our results revealed that PTD-CcTrx1 exhibited specific transmembrane ability and antioxidant activities, and it could significantly attenuate the intracellular oxidative stress, inhibit H2O2-induced apoptosis, and protect HaCaT cells from oxidative damage. The present study provides critical evidence for application of PTD-CcTrx1 as a novel antioxidant to treat skin oxidative damage in the future.

HIV-1 Tat-mediated microglial ferroptosis involves the miR-204-ACSL4 signaling axis.

This study was focused on exploring the role of the HIV-1 Tat protein in mediating microglial ferroptosis. Exposure of mouse primary microglial cells (mPMs) to HIV-1 Tat protein resulted in induction of ferroptosis, which was characterized by increased expression of Acyl-CoA synthetase long-chain family member 4 (ACSL4), in turn, leading to increased generation of oxidized phosphatidylethanolamine, elevated levels of lipid peroxidation, upregulated labile iron pool (LIP) and ferritin heavy chain-1 (FTH1), decreased glutathione peroxidase-4 and mitochondrial outer membrane rupture. Also, inhibition of ferroptosis by ferrostatin-1 (Fer-1) or deferoxamine (DFO) treatment suppressed ferroptosis-related changes in mPMs. Similarly, the knockdown of ACSL4 by gene silencing also inhibited ferroptosis induced by HIV-1 Tat. Furthermore, increased lipid peroxidation resulted in increased release of proinflammatory cytokines, such as TNFα, IL6, and IL1ß and microglial activation. Pretreatment of mPMs with Fer-1 or DFO further blocked HIV-1 Tat-mediated microglial activation in vitro and reduced the expression and release of proinflammatory cytokines. We identified miR-204 as an upstream modulator of ACSL4, which was downregulated in mPMs exposed to HIV-1 Tat. Transient transfection of mPMs with miR-204 mimics reduced the expression of ACSL4 while inhibiting HIV-1 Tat-mediated ferroptosis and the release of proinflammatory cytokines. These in vitro findings were further validated in HIV-1 transgenic rats as well as HIV + ve human brain samples. Overall, this study underscores a novel mechanism(s) underlying HIV-1 Tat-mediated ferroptosis and microglial activation involving miR-204-ACSL4 signaling.

Disruption of Mitochondrial-associated ER membranes by HIV-1 tat protein contributes to premature brain aging.

INTRODUCTION: Mitochondrial-associated ER membranes (MAMs) control many cellular functions, including calcium and lipid exchange, intracellular trafficking, and mitochondrial biogenesis. The disruption of these functions contributes to neurocognitive disorders, such as spatial memory impairment and premature brain aging. Using neuronal cells, we demonstrated that HIV-1 Tat protein deregulates the mitochondria. METHODS& RESULTS: To determine the mechanisms, we used a neuronal cell line and showed that Tat-induced changes in expression and interactions of both MAM-associated proteins and MAM tethering proteins. The addition of HIV-1 Tat protein alters expression levels of PTPIP51 and VAPB proteins in the MAM fraction but not the whole cell. Phosphorylation of PTPIP51 protein regulates its subcellular localization and function. We demonstrated that the Tat protein promotes PTPIP51 phosphorylation on tyrosine residues and prevents its binding to VAPB. Treatment of the cells with a kinase inhibitor restores the PTPIP51-VAPB interaction and overcomes the effect of Tat. CONCLUSION: These results suggest that Tat disrupts the MAM, through the induction of PTPIP51 phosphorylation, leading to ROS accumulation, mitochondrial stress, and altered movement. Hence, we concluded that interfering in the MAM-associated cellular pathways contributes to spatial memory impairment and premature brain aging often observed in HIV-1-infected patients.

Tat-Thioredoxin-like protein 1 attenuates ischemic brain injury by regulation of MAPKs and apoptosis signaling.

Thioredoxin-like protein 1 (TXNL1), one of the thioredoxin superfamily known as redox-regulator, plays an essential in maintaining cell survival via various antioxidant and anti-apoptotic mechanisms. It is well known that relationship between ischemia and oxidative stress, however, the role of TXNL1 protein in ischemic damage has not been fully investigated. In the present study, we aimed to determine the protective role of TXNL1 against on ischemic injury in vitro and in vivo using cell permeable Tat-TXNL1 fusion protein. Transduced Tat-TXNL1 inhibited ROS production and cell death in H2O2-exposed hippocampal neuronal (HT-22) cells and modulated MAPKs and Akt activation, and pro-apoptotic protein expression levels in the cells. In an ischemia animal model, Tat-TXNL1 markedly decreased hippocampal neuronal cell death and the activation of astrocytes and microglia. These findings indicate that cell permeable Tat-TXNL1 protects against oxidative stress in vitro and in vivo ischemic animal model. Therefore, we suggest Tat-TXNL1 can be a potential therapeutic protein for ischemic injury. [BMB Reports 2023; 56(4): 234-239].

Knockout of Tyrosine Aminotransferase Gene by Homologous Recombination Arrests Growth and Disrupts Redox Homeostasis in Leishmania Parasite.

Tyrosine aminotransferase is a well-characterized enzyme in the Leishmania parasite, but the role of TAT in the parasite functioning remains largely unknown. In this study, we attempt to gain a better understanding of the enzyme's role in the parasite by gene knockout and overexpression of the TAT gene. The overexpression of TAT protein was well tolerated by the parasites in two independent repeats. Single knockout of TAT gene by homologous recombination, LdTAT+/- displayed distinct retardation in the proliferation rates and entered the death phase immediately. Morphology of LdTAT+/- parasites had important structural defects as they rounded up with elongated flagella. Gene regulation studies suggested the upregulation of key apoptotic and redox metabolism genes in LdTAT+/-. Moreover, LdTAT+/- cells accumulated higher ROS, thiols, intracellular Ca2+ concentrations, and mitochondrial membrane depolarization signifying the onset of apoptosis. Tocopherol levels were reduced by 50% in LdTAT+/- suggesting the involvement of TAT in tocopherol biosynthesis in the parasite. Overall, our results provide the first evidence that gene knockout of TAT results in apoptosis and that TAT is required for the survival and viability of Leishmania donovani.

HIV-1 Tat Protein-Mediated Inflammatory Response Inhibits the Erythroid Hematopoietic Support Function of Bone Marrow Mesenchymal Stem Cells.

Although combination antiretroviral therapy is widely used to treat HIV-1 infection, anemia affects the health and quality of life in a large number of these patients. The proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs), as important support cells in the hematopoietic microenvironment, can be affected by HIV-1 Tat protein. In this study, we explored the mechanism underlying the effect of Tat protein on the hematopoietic support function of BMSCs in erythroid commitment. BMSCs were treated with Tat protein or transfected with Tat mRNA and cocultured with hematopoietic stem cells (HSCs) to detect the number of erythroid colony-forming units (CFUs) and the proportion of mature red blood cells from HSCs. Subsequently, the expression level of a series of erythroid hematopoietic support factors and inflammatory factors in BMSCs after Tat treatment were analyzed. Then, the activation effect of Tat on the mitogen-activated protein kinase/nuclear factor kappa-B (MAPK/NF-κB) pathway, which is an important inflammatory response signaling pathway, was evaluated. The results showed that the number of erythroid CFUs and the production of mature red blood cells supported by BMSCs treated with Tat protein were significantly reduced and the expression of a series of erythroid supporting factors of BMSCs were significantly decreased by Tat protein. Tat-treated BMSCs highly express a variety of inflammatory mediators. Moreover, the expression of P38, p-p38, ERK1/2, p-ERK1/2, JNK1/2, p-JNK1/2, NF-κB, and p-NF-κB was significantly upregulated by Tat protein. In conclusion, Tat protein induces the inflammatory response of BMSCs by activating the MAPK/NF-κB pathway to inhibit the erythroid hematopoietic support function of BMSCs.

Tfeb-Mediated Transcriptional Regulation of Autophagy Induces Autosis during Ischemia/Reperfusion in the Heart.

Autosis is a unique form of cell death with characteristic morphological and biochemical features caused by dysregulated autophagy. Autosis is observed in the heart during the late phase of ischemia/reperfusion (I/R), when marked accumulation of autophagosomes is induced. We previously showed that the excessive accumulation of autophagosomes promotes autosis in cardiomyocytes. Although the inhibition of autophagic flux via the upregulation of Rubicon induces the accumulation of autophagosomes during I/R, it appears that additional mechanisms exacerbating autophagosome accumulation are required for the induction of autosis. Here, we show that Tfeb contributes to the induction of autosis during the late phase of I/R in the heart. During myocardial reperfusion, Tfeb is activated and translocated into the nucleus, which in turn upregulates genes involved in autophagy and lysosomal function. The overexpression of Tfeb enhanced cardiomyocyte death induced by a high dose of TAT-Beclin 1, an effect that was inhibited by the downregulation of Atg7. Conversely, the knockdown of Tfeb attenuated high-dose TAT-Beclin1-induced death in cardiomyocytes. Although the downregulation of Tfeb in the heart significantly decreased the number of autophagic vacuoles and inhibited autosis during I/R, the activation of Tfeb activity via 3,4-dimethoxychalcone, an activator of Tfeb, aggravated myocardial injury during I/R. These findings suggest that Tfeb promotes cardiomyocyte autosis during the late phase of reperfusion in the heart.

TAT-RHIM: a more complex issue than expected.

Murine cytomegalovirus protein M45 contains a RIP homotypic interaction motif (RHIM) that is sufficient to confer protection of infected cells against necroptotic cell death. Mechanistically, the N-terminal region of M45 drives rapid self-assembly into homo-oligomeric amyloid fibrils, and interacts with the endogenous RHIM domains of receptor-interacting serine/threonine protein kinases (RIPK) 1, RIPK3, Z-DNA-binding protein 1, and Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-ß. Remarkably, all four aforementioned mammalian proteins harbouring such a RHIM domain are key components of inflammatory signalling and regulated cell death (RCD) processes. Immunogenic cell death by regulated necrosis causes extensive tissue damage in a wide range of diseases, including ischaemia reperfusion injury, myocardial infarction, sepsis, stroke, and solid organ transplantation. To harness the cell death suppression properties of M45 protein in a therapeutically usable manner, we developed a synthetic peptide encompassing only the RHIM domain of M45. To trigger delivery of RHIM into target cells, we fused the transactivator protein transduction domain of human immunodeficiency virus 1 to the N-terminus of the peptide. The fused peptide could efficiently penetrate eukaryotic cells, but unexpectedly it eradicated or destroyed all tested cancer cell lines and primary cells irrespective of species without further stimulus through a necrosis-like cell death. Typical inhibitors of different forms of RCD cannot impede this process, which appears to involve a direct disruption of biomembranes. Nevertheless, our finding has potential clinical relevance; reliable induction of a necrotic form of cell death distinct from all known forms of RCD may offer a novel therapeutic approach to combat resistant tumour cells.

Therapeutic Intervention Using a Smad7-Based Tat Protein to Treat Radiation-Induced Oral Mucositis.

PURPOSE: Recent studies reported therapeutic effects of Smad7 on oral mucositis in mice without compromising radiation therapy-induced cancer cell killing in neighboring oral cancer. This study aims to assess whether a Smad7-based biologic can treat oral mucositis in a clinically relevant setting by establishing an oral mucositis model in dogs and analyzing molecular targets. METHODS AND MATERIALS: We created a truncated human Smad7 protein fused with the cell-penetrating Tat tag (Tat-PYC-Smad7). We used intensity modulated radiation therapy to induce oral mucositis in dogs and applied Tat-PYC-Smad7 to the oral mucosa in dose-finding studies after intensity modulated radiation therapy. Clinical outcomes were evaluated. Molecular targets were analyzed in biopsies and serum samples. RESULTS: Tat-PYC-Smad7 treatment significantly shortened the duration of grade 3 oral mucositis based on double-blinded Veterinary Radiation Therapy Oncology Group scores and histopathology evaluations. Topically applied Tat-PYC-Smad7 primarily penetrated epithelial cells and was undetectable in serum. NanoString nCounter Canine IO Panel identified that, compared to the vehicle samples, top molecular changes in Tat-PYC-Smad7 treated samples include reductions in inflammation and cell death and increases in cell growth and DNA repair. Consistently, immunostaining shows that Tat-PYC-Smad7 reduced DNA damage and neutrophil infiltration with attenuated TGF-ß and NFκB signaling. Furthermore, IL-1ß and TNF-α were lower in Tat-PYC-Smad7 treated mucosa and serum samples compared to those in vehicle controls. CONCLUSIONS: Topical Tat-PYC-Smad7 application demonstrated therapeutic effects on oral mucositis induced by intensity modulated radiation therapy in dogs. The local effects of Tat-PYC-Smad7 targeted molecules involved in oral mucositis pathogenesis as well as reduced systemic inflammatory cytokines.

Characterization of the membrane penetration-enhancing peptide S19 derived from human syncytin-1 for the intracellular delivery of TAT-fused proteins.

Although cell-penetrating peptides such as the HIV-derived TAT peptide have been used as tools for the intracellular delivery of therapeutic peptides and proteins, a problem persists: the endosomal escape efficiency is low. Previously, we found that the fusogenic peptide S19, derived from the human protein syncytin-1, enhance the endosomal escape efficiency of proteins that incorporated by endocytosis via TAT. In this study, we first performed Ala-scanning mutagenesis of S19, and found that all Ile, Val, Leu and Phe with high ß-sheet forming propensities in S19 are important for the intracellular uptake of S19-TAT-fused proteins. In a secondary structure analysis of the mutated S19-TAT peptides in the presence of liposomes mimicking late endosomes (LEs), the CD spectra of V3A and I4A mutants with low uptake activity showed the appearance of an α-helix structure, whereas the mutant G5A retained both the uptake activity and the ß-structure. In addition, we investigated the appropriate linking position and order of the S19 and TAT peptides to a cargo protein including an apoptosis-induced peptide and found that both the previous C-terminal S19-TAT tag and the N-terminal TAT-S19 tag promote the cytoplasmic delivery of the fusion protein. These results and previous results suggest that the interaction of TAT with the LE membrane causes a structural change in S19 from a random coil to a ß-strand and that the subsequent parallel ß-sheet formation between two S19 peptides may promote adjacent TAT dimerization, resulting in endosomal escape from the LE membrane.

Antioxidant Fusion Protein SOD1-Tat Increases the Engraftment Efficiency of Total Bone Marrow Cells in Irradiated Mice.

Total body irradiation is a standard procedure of bone marrow transplantation (BMT) which causes a rapid increase in reactive oxygen species (ROS) in the bone marrow microenvironment during BMT. The increase in ROS reduces the engraftment ability of donor cells, thereby affecting the bone marrow recovery of recipients after BMT. In the early weeks following transplantation, recipients are at high risk of severe infection due to weakened hematopoiesis. Thus, it is imperative to improve engraftment capacity and accelerate bone marrow recovery in BMT recipients. In this study, we constructed recombinant copper/zinc superoxide dismutase 1 (SOD1) fused with the cell-penetrating peptide (CPP), the trans-activator of transcription (Tat), and showed that this fusion protein has penetrating ability and antioxidant activity in both RAW264.7 cells and bone marrow cells in vitro. Furthermore, irradiated mice transplanted with SOD1-Tat-treated total bone marrow donor cells showed an increase in total bone marrow engraftment capacity two weeks after transplantation. This study explored an innovative method for enhancing engraftment efficiency and highlights the potential of CPP-SOD1 in ROS manipulation during BMT.

Tat-CIAPIN1 protein prevents against cytokine-induced cytotoxicity in pancreatic RINm5F ß-cells.

Cytokines activate inflammatory signals and are major mediators in progressive ß-cell damage, which leads to type 1 diabetes mellitus. We recently showed that the cell-permeable Tat-CIAPIN1 fusion protein inhibits neuronal cell death induced by oxidative stress. However, how the Tat-CIAPIN1 protein affects cytokine-induced ß-cell damage has not been investigated yet. Thus, we assessed whether the Tat-CIAPIN1 protein can protect RINm5F ß-cells against cytokine-induced cytotoxicity. In cytokine-exposed RINm5F ß-cells, the transduced Tat-CIAPIN1 protein elevated cell survivals and reduced reactive oxygen species (ROS) and DNA fragmentation levels. The Tat-CIAPIN1 protein reduced mitogen-activated protein kinases (MAPKs) and NF-κB activation levels and elevated Bcl-2 protein, whereas Bax and cleaved Caspase-3 proteins were decreased by this fusion protein. Thus, the protection of RINm5F ß-cells by the Tat-CIAPIN1 protein against cytokine-induced cytotoxicity can suggest that the Tat-CIAPIN1 protein might be used as a therapeutic inhibitor against RINm5F ß-cell damage. [BMB Reports 2021; 54(9): 458-463].

Electrochromic shift supports the membrane destabilization model of Tat-mediated transport and shows ion leakage during Sec transport.

The mechanism and pore architecture of the Tat complex during transport of folded substrates remain a mystery, partly due to rapid dissociation after translocation. In contrast, the proteinaceous SecY pore is a persistent structure that needs only to undergo conformational shifts between "closed" and "opened" states when translocating unfolded substrate chains. Where the proteinaceous pore model describes the SecY pore well, the toroidal pore model better accounts for the high-energy barrier that must be overcome when transporting a folded substrate through the hydrophobic bilayer in Tat transport. Membrane conductance behavior can, in principle, be used to distinguish between toroidal and proteinaceous pores, as illustrated in the examination of many antimicrobial peptides as well as mitochondrial Bax and Bid. Here, we measure the electrochromic shift (ECS) decay as a proxy for conductance in isolated thylakoids, both during protein transport and with constitutively assembled translocons. We find that membranes with the constitutively assembled Tat complex and those undergoing Tat transport display conductance characteristics similar to those of resting membranes. Membranes undergoing Sec transport and those with the substrate-engaged SecY pore result in significantly more rapid electric field decay. The responsiveness of the ECS signal in membranes with active SecY recalls the steep relationship between applied voltage and conductance in a proteinaceous pore, while the nonaccelerated electric field decay with both Tat transport and the constitutive Tat complex under the same electric field is consistent with the behavior of a toroidal pore.

Tat-Endophilin A1 Fusion Protein Protects Neurons from Ischemic Damage in the Gerbil Hippocampus: A Possible Mechanism of Lipid Peroxidation and Neuroinflammation Mitigation as Well as Synaptic Plasticity.

The present study explored the effects of endophilin A1 (SH3GL2) against oxidative damage brought about by H2O2 in HT22 cells and ischemic damage induced upon transient forebrain ischemia in gerbils. Tat-SH3GL2 and its control protein (Control-SH3GL2) were synthesized to deliver it to the cells by penetrating the cell membrane and blood-brain barrier. Tat-SH3GL2, but not Control-SH3GL2, could be delivered into HT22 cells in a concentration- and time-dependent manner and the hippocampus 8 h after treatment in gerbils. Tat-SH3GL2 was stably present in HT22 cells and degraded with time, by 36 h post treatment. Pre-incubation with Tat-SH3GL2, but not Control-SH3GL2, significantly ameliorated H2O2-induced cell death, DNA fragmentation, and reactive oxygen species formation. SH3GL2 immunoreactivity was decreased in the gerbil hippocampal CA1 region with time after ischemia, but it was maintained in the other regions after ischemia. Tat-SH3GL2 treatment in gerbils appreciably improved ischemia-induced hyperactivity 1 day after ischemia and the percentage of NeuN-immunoreactive surviving cells increased 4 days after ischemia. In addition, Tat-SH3GL2 treatment in gerbils alleviated the increase in lipid peroxidation as assessed by the levels of malondialdehyde and 8-iso-prostaglandin F2α and in pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1ß, and interleukin-6; while the reduction of protein levels in markers for synaptic plasticity, such as postsynaptic density 95, synaptophysin, and synaptosome associated protein 25 after transient forebrain ischemia was also observed. These results suggest that Tat-SH3GL2 protects neurons from oxidative and ischemic damage by reducing lipid peroxidation and inflammation and improving synaptic plasticity after ischemia.