Tat Peptide Research Articles

Guanidines Conjugated with Cell-Penetrating Peptides: A New Approach for the Development of Antileishmanial Molecules

Leishmaniasis is a neglected tropical disease caused by a protozoan of the genus Leishmania, which has visceral and cutaneous forms. The symptoms of leishmaniasis include high fever and weakness, and the cutaneous infection also causes lesions under the skin. The drugs used to treat leishmaniasis have become less effective due to the resistance mechanisms of the protozoa. In addition, the current compounds have low selectivity for the pathogen, leading to various side effects, which results in lower adherence to treatment. Various strategies were developed to solve this problem. The bioconjugation between natural compounds with antimicrobial activity and cell-penetrating peptides could alleviate the resistance and toxicity of current treatments. This work aims to conjugate the cell penetration peptide TAT to the guanidine GVL1. The GVL1-TAT bioconjugate exhibited leishmanicidal activity against Leishmania amazonensis and Leishmania infantum with a high selectivity index. In addition, the bioconjugate was more active against the intracellular enzyme CPP than the individual compounds. This target is very important for the viability and virulence of the parasite within the host cell. Docking studies confirmed the higher interaction of the conjugate with CPP and suggested that other proteins, such as trypanothione reductase, could be targeted. Thus, the data indicated that guanidines conjugated with cell-penetrating peptides could be a good approach for developing antileishmanial molecules.

Transduction of jellyfish superoxide dismutase mediated by TAT peptide ameliorates H2O2-induced oxidative stress in HaCaT cells

Superoxide dismutase (SOD) plays important roles in the balance of oxidation and antioxidation in body mostly by scavenging superoxide anion free radicals (O2.−). Previously, we reported a novel Cu/Zn SOD from jellyfish Cyanea capillata, named CcSOD1, which exhibited excellent SOD activity and high stability. TAT peptide is a common type of cell penetrating peptides (CPPs) that efficiently deliver extracellular biomacromolecules into cytoplasm. In this study, we constructed a recombinant expression vector that combined the coding sequences of TAT peptide and CcSOD1, and then obtained sufficient and high-purity TAT-CcSOD1 fusion protein. Compared with some reported SODs/CPP-SODs, TAT-CcSOD1 possessed stronger tolerance to heat and acid–base environment. TAT-CcSOD1 efficiently penetrated cell membrane and significantly enhanced the O2.− scavenging ability in cells, and attenuated H2O2-induced cytotoxicity and NO generation in HaCaT cells. This study serves as a critical step forward for the application of TAT-CcSOD1 as a potential protective/therapeutic agent against oxidative stress-related conditions in the future.

In vivo scanning laser fundus and high-resolution OCT imaging of retinal ganglion cell injury in a non-human primate model with an activatable fluorescent-labeled TAT peptide probe.

The optical imaging agent TcapQ488 has enabled imaging of retinal ganglion cell (RGC) injury in vivo in rodents and has potential as an effective diagnostic probe for early detection and intervention monitoring in glaucoma patients. In the present study, we investigated TcapQ488 in non-human primates (NHPs) to identify labeling efficacy and early signals of injured RGC, to determine species-dependent changes in RGC probe uptake and clearance, and to determine dose-limiting toxicities. Doses of 3, 6, and 12 nmol of TcapQ488 were delivered intravitreally to normal healthy NHP eyes and eyes that had undergone hemiretinal endodiathermy axotomy (HEA) in the inferior retina. Post-injection fundus fluorescence imaging using a Spectralis imaging platform (Heidelberg Engineering) documented TcapQ488 activation in RGC cell bodies. Optical coherence tomography (OCT), slit-lamp examinations, intraocular pressure measurements, and visual electrophysiology testing were performed to monitor probe tolerability. For comparison, a negative control, non-cleavable, non-quenched probe (dTcap488, 6 nmol), was delivered intravitreally to a normal healthy eye. In normal healthy eyes, intravitreal injection of 3 nmol of TcapQ488 was well-tolerated, while 12 nmol of TcapQ488 to the healthy eye caused extensive probe activation in the ganglion cell layer (GCL) and eventual retinal nerve fiber layer thinning. In HEA eyes, the HEA procedure followed by intravitreal TcapQ488 (3 nmol) injection resulted in probe activation within cell bodies in the GCL, confined to the HEA-treated inferior retina, indicating cell injury and slow axonal transport in the GCL. However, in contrast to rodents, a vitreal haze that lasted 2-12 weeks obscured rapid high-resolution imaging of the fundus. By contrast, intravitreal TcapQ488 injection prior to the HEA procedure led to minimal probe labeling in the GCL. The results of the dTcap488 control experiments indicated that fast axonal transport carried the probe out of the retina after cell body uptake. No evidence of pan-retinal toxicity or loss of retino-cortical function was detected in any of the three NHPs tested. Overall, these data provide evidence of TcapQ488 activation, without toxicity, in NHP HEA eyes that had been intravitreally injected with 3 nmol of the probe. Compared to rodents, unexpectedly rapid axonal transport in the NHPs reduced the capacity to visualize RGC cell bodies and axons through the backdrop of an intravitreal haze. Nonetheless, although intravitreal clearance rates did not scale to NHPs, HEA-induced reductions in axonal transport enhanced probe visualization in the cell body.

Engineering a nano-drug delivery system to regulate m6A modification and enhance immunotherapy in gastric cancer

Cancer cell membrane-derived nanoparticle drug delivery system enables precise drug delivery to tumor tissues and is a new effective way to treat solid tumors. The aim of this study is to develop a safe and effective cancer cell membrane-derived nano-delivery system targeting gastric cancer. We previously reported that EPH receptor A2 (EphA2) is an important target for gastric cancer. RNA m6A methyltransferases METTL3 is upregulated in multiple cancers and promotes cancer development by increasing the expression of multiple oncogenes. We design a new nano-delivery system PLGA-STM-TAT: nanoparticles PLGA (poly lactic acid-hydroxyacetic acid) loaded with METTL3 inhibitor STM2457 and cell-penetrating peptide TAT, and then covered with gastric cancer cell membranes equipped with YSA peptides by means of click chemistry, which targeting EphA2. The nanoparticles are specifically enriched in gastric cancer tissues, significantly increased drug accumulation, and inhibited cancer cell proliferation by decreasing key oncogenes c-MYC and BRD4. During drug administration, we found that the expression of the immune checkpoint molecule PD-L1 was suppressed, and the anti-tumor immune effect was enhanced by the nano-delivery system in combination with anti-PD1. This cancer cell membrane-derived nano-delivery system provides a new biological strategy to treat gastric cancer through effective m6A modulation and EphA2 targeting. Statement of significanceM6A modifications have important biological roles, especially in tumors. Targeting highly modified m6A in gastric cancer becomes a challenge. We developed a nano-drug delivery system for modulating m6A that could produce an effective anti-cancer therapeutic effect and that the nanoparticles enhanced antitumor immunity when combined with anti-PD1.This cancer cell membrane-derived new nano-drug delivery system shows great promise as an antitumor approach by modulating m6A modification and targeting EphA2 in gastric cancers.

Insights into Translocation of Arginine-Rich Cell-Penetrating Peptides across a Model Membrane.

It is well-known that membrane deformation and water pores contribute to the spontaneous translocation of arginine-rich cell-penetrating peptides (CPPs). We confirm this through the observation of the spontaneous translocation of single R9 (nona-arginine) and Tat (48-60) peptides across a model membrane using the weighted ensemble (WE) method within all-atom molecular dynamics (MD) simulations. Furthermore, we demonstrate that membrane deformation and the presence of a water pore reduce the effective charge of the CPP and the bending rigidity of the model membrane during translocation. We find that R9 disturbs the model membrane more than Tat (48-60), leading to more efficient translocation of R9 across the model membrane.

MeCP2 is a naturally supercharged protein with cell membrane transduction capabilities.

The intrinsically disordered protein MeCP2 is a global transcriptional regulator encoded by the MECP2 gene. Although the structured domains of MeCP2 have been the subject of multiple studies, its unstructured regions have not been that extensively characterized. In this work, we show that MeCP2 possesses properties akin to those of supercharged proteins. By utilizing its unstructured portions, MeCP2 can successfully transduce across cell membranes and localize to heterochromatic foci in the nuclei, displaying uptake levels a third lower than a MeCP2 construct fused to the cell-penetrating peptide TAT. MeCP2 uptake can further be enhanced by the addition of compounds that promote endosomal escape following cellular trafficking by means of macropinocytosis. Using a combination of in silico prediction algorithms and live-cell imaging experiments, we mapped the sequence in MeCP2 responsible for its cellular incorporation, which bears a striking resemblance to TAT itself. Transduced MeCP2 was shown to interact with HDAC3. These findings provide valuable insight into the properties of MeCP2 and may be beneficial for devising future protein-based treatment strategies.

Investigation of Dual-Loaded Doxorubicin and Sorafenib Liposomes Co-Modified with Glycyrrhetinic Acid and Cell-Penetrating Peptide TAT.

Combining Doxorubicin (DOX) with sorafenib (SF) is a promising strategy for treating Hepatocellular Carcinoma (HCC). However, strict dosage control is required for both drugs, and there is a lack of target selectivity. This study aims to develop a novel nano-drug delivery system for the combined use of DOX and SF, aiming to reduce their respective dosages, enhance therapeutic efficacy, and improve target selectivity. DOX/SF co-loaded liposomes (LPs) were prepared using the thin-film hydration method. The liposomes were modified with 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine (DSPE)- polyethylene glycol (PEG2000), DSPE-PEG1000-cell penetrating peptide TAT, and Glycyrrhetinic Acid (GA). The basic properties of the liposomes were characterized. CCK-8 cell viability assays were conducted using HepG2, MHCC97-H, and PLC cell models, and apoptosis experiments were performed using HepG2 cells to determine if this delivery system could reduce the respective dosages of DOX and SF and enhance HCC cytotoxicity. Liposome uptake experiments were performed using HepG2 cells to validate the target selectivity of this delivery system. A GA/TAT-DOX/SF-LP liposomal nano drug delivery system was successfully constructed, with a particle size of 150 nm, a zeta potential of -7.9 mV, a DOX encapsulation efficiency of 92%, and an SF encapsulation efficiency of 88.7%. Cellular experiments demonstrated that this delivery system reduced the required dosages of DOX and SF, exhibited stronger cytotoxicity against liver cancer cells, and showed better target selectivity. A simple and referenceable liposomal nano drug delivery system has been developed for the combined application of DOX and SF in hepatocellular carcinoma treatment.

Cell-penetrating peptides TAT and 8R functionalize P22 virus-like particles to enhance tissue distribution and retention in vivo.

Virus-like particles (VLPs) are used as nanocontainers for targeted drug, protein, and vaccine delivery. The phage P22 VLP is an ideal macromolecule delivery vehicle, as it has a large exterior surface area, which facilitates multivalent genetic and chemical modifications for cell recognition and penetration. Arginine-rich cell-penetrating peptides (CPPs) can increase cargo transport efficiency in vivo. However, studies on the tissue distribution and retention of P22 VLPs mediated by TAT and 8R are lacking. This study aimed to analyze the TAT and 8R effects on the P22 VLPs transport efficiency and tissue distribution both in vitro and in vivo. We used a prokaryotic system to prepare P22 VLP self-assembled particles and expressed TAT-or 8R-conjugated mCherry on the VLP capsid protein as model cargoes and revealed that the level of P22 VLP-mCherry penetrating the cell membrane was low. However, both TAT and 8R significantly promoted the cellular uptake efficiency of P22 VLPs in vitro, as well as enhanced the tissue accumulation and retention of P22 VLPs in vivo. At 24 h postinjection, TAT enhanced the tissue distribution and retention in the lung, whereas 8R could be better accumulation in brain. Thus, TAT was superior in terms of cellular uptake and tissue accumulation in the P22 VLPs delivery system. Understanding CPP biocompatibility and tissue retention will expand their potential applications in macromolecular cargo delivery.

Design of deep-red emissive forced intercalation-induced light-up peptide as an indicator for the HIV-1 TAR RNA-ligand assay: integration of benzo[c,d]indole-quinoline (BIQ) cyanine dye into Tat peptide.

We report on a deep-red emissive fluorogenic peptide probe for human immunodeficiency virus-1 (HIV-1) trans-activation responsive (TAR) RNA as an indicator for fluorescence indicator displacement (FID) assay. The probe design is based on the concept of the forced intercalation of thiazole orange (TO) dyes (FIT) on the peptide backbone, as recently proposed by our group, where the Q (glutamic acid) residue in the Tat peptide (RKKRR-Q-RRR) is replaced with TO as if it were an amino acid surrogate. Here, instead of green emissive TO, we utilized a deep-red emissive benzo[c,d]indole-quinoline (BIQ) cyanine dye developed previously by our group for imaging of nucleolar RNA in living cells. The developed 9-mer FIT peptide (RKKRR-BIQ-RRR; named BIQ-FiLuP) exhibits a significant off-on signaling ability for TAR RNA (λem = 660nm, I/I0 = 130-fold, Φfree = 0.0009, Φbound = 0.052), and the dissociation constant Kd reaches ca. 1nM. When used in FID assay, BIQ-FiLuP, like TO-based FiLuP, is able to distinguish between competitive and noncompetitive inhibitors, which has never been demonstrated with all previous indicators for TAR RNA. Deep-red emissive BIQ-FiLuP facilitates the evaluation of green to yellow emissive ligands without suffering from optical interference. The combination use with green emissive TO-based FiLuP (λem = 541nm) would cover the examination of a wide range of fluorescent test compounds.

Expression, purification and application of a recombinant, membrane permeating version of the light chain of botulinum toxin B.

Botulinum neurotoxins (BoNTs) are valuable tools to unveil molecular mechanisms of exocytosis in neuronal and non-neuronal cells due to their peptidase activity on exocytic isoforms of SNARE proteins. They are produced by Clostridia as single-chain polypeptides that are proteolytically cleaved into light, catalytic domains covalently linked via disulfide bonds to heavy, targeting domains. This format of two subunits linked by disulfide bonds is required for the full neurotoxicity of BoNTs. We have generated a recombinant version of BoNT/B that consists of the light chain of the toxin fused to the protein transduction domain of the human immunodeficiency virus-1 (TAT peptide) and a hexahistidine tag. His6-TAT-BoNT/B-LC, expressed in Escherichia coli and purified by affinity chromatography, penetrated membranes and exhibited strong enzymatic activity, as evidenced by cleavage of the SNARE synaptobrevin from rat brain synaptosomes and human sperm cells. Proteolytic attack of synaptobrevin hindered exocytosis triggered by a calcium ionophore in the latter. The novel tool reported herein disrupts the function of a SNARE protein within minutes in cells that may or may not express the receptors for the BoNT/B heavy chain, and without the need for transient transfection or permeabilization.

WITHDRAWN: Folic acid Grafted, cell-penetrating peptide conjugated pH-sensitive hydrophobically modified Glycol chitosan nanoparticles for drug delivery in breast cancer

Nanocarriers are an outcome of the fast development of nanotechnology; however, delivering drugs to tumor sites effectively is still a major difficulty. A pH-responsive drug carrier with good cell penetration and targeting ability was developed to release the drug exactly to the cancer site and overcome these limitations.The drug carrier FA-TAT-HGCNPs/PTX, which is pH-responsive, uses folic acid (FA) and TAT peptide to deliver paclitaxel (PTX) for target folate-receptor-overexpressing breast cancer cells. 2-(diisopropylamino) ethyl methacrylate was connected to glycol chitosan (GC) to form HGCNPs. Then TAT and HGCNPs were conjugated and FA was grafted onto the HGCNPs’ hydroxyl group. Physicochemical characterizations of FA-TAT-HGCNPs was also evaluated. The maximum DLE and EE values of the FA-TAT-HGCNPs at pH 8.0 were 74.0 ± 2.22 % and 93.7 ± 2.8 %, respectively. FA-TAT-HGCNPs, for instance, showed stability during long-term storage and lyophilization while exhibiting pH-responsive dissociation properties. The synthesized drug carrier was characterized by FTIR, XRD, SEM, HRTEM, DLS, and Zeta analysis. MTT assay exhibited the high anticancer activity of the FA-TAT-HGCNPs/PTX against the MCF-7 cells, whereas drug-free ones are highly biocompatible. The pharmacokinetic results showed twice the bioavailability of FA-TAT-HGCNPs/PTX compared to HGCNPs/PTX. Unlike FA-HGCNPs/PTX, CLSM analysis showed that FA-TAT-HGCNPs/PTX had a significantly more efficient intracellular uptake in MCF-7 cells. These data have shown the transmembrane ability of TATpeptide,the targeting effects of folate decoration, and the synergistic interaction between them.According to these findings, PTX-loaded FA-TAT-HGCNPs offer a potentially effective nanoscale drug delivery system for the treatment of breast cancer.

CRISPR/Pepper-tDeg: A Live Imaging System Enables Non-Repetitive Genomic Locus Analysis with One Single-Guide RNA.

CRISPR-based genomic-imaging systems have been utilized for spatiotemporal imaging of the repetitive genomic loci in living cells, but they are still challenged by limited signal-to-noise ratio (SNR) at a non-repetitive genomic locus. Here, an efficient genomic-imaging system is proposed, termed CRISPR/Pepper-tDeg, by engineering the CRISPR sgRNA scaffolds with the degron-binding Pepper aptamers for binding fluorogenic proteins fused with Tat peptide derived degron domain (tDeg). The target-dependent stability switches of both sgRNA and fluorogenic protein allow this system to image repetitive telomeres sensitively with a 5-fold higher SNR than conventional CRISPR/MS2-MCP system using "always-on" fluorescent protein tag. Subsequently, CRISPR/Pepper-tDeg is applied to simultaneously label and track two different genomic loci, telomeres and centromeres, in living cells by combining two systems. Given a further improved SNR by the split fluorescent protein design, CRISPR/Pepper-tDeg system is extended to non-repetitive sequence imaging using only one sgRNA with two aptamer insertions. Neither complex sgRNA design nor difficult plasmid construction is required, greatly reducing the technical barriers to define spatiotemporal organization and dynamics of both repetitive and non-repetitive genomic loci in living cells, and thus demonstrating the large application potential of this genomic-imaging system in biological research, clinical diagnosis and therapy.

IL-37d suppresses Rheb-mTORC1 axis independently of TCS2 to alleviate alcoholic liver disease

Tuberous sclerosis complex 2 (TSC2) crucially suppresses Rheb activity to prevent mTORC1 activation. However, mutations in TSC genes lead to mTORC1 overactivation, thereby causing various developmental disorders and cancer. Therefore, the discovery of novel Rheb inhibitors is vital to prevent mTOR overactivation. Here, we reveals that the anti-inflammatory cytokine IL-37d can bind to lysosomal Rheb and suppress its activity independent of TSC2, thereby preventing mTORC1 activation. The binding of IL-37d to Rheb switch-II subregion destabilizes the Rheb-mTOR and mTOR-S6K interactions, further halting mTORC1 signaling. Unlike TSC2, IL-37d is reduced under ethanol stimulation, which results in mitigating the suppression of lysosomal Rheb-mTORC1 activity. Consequently, the recombinant human IL-37d protein (rh-IL-37d) with a TAT peptide greatly improves alcohol-induced liver disorders by hindering Rheb-mTORC1 axis overactivation in a TSC2- independent manner. Together, IL-37d emerges as a novel Rheb suppressor independent of TSC2 to terminate mTORC1 activation and improve abnormal lipid metabolism in the liver.

Unlocking Therapeutic Potential: Enhanced shRNA Delivery with Tat Peptide in the Human Respiratory Syncytial Virus Treatment.

This research investigated the development of short hairpin RNA (shRNA) molecules designed to target specific regions of the human respiratory syncytial virus (HRSV) M and F genes. The study aimed to assess the therapeutic potential of these shRNAs and evaluate the effectiveness of Tat peptide-mediated delivery in enhancing their functionality. We acquired isolates from pediatric patients experiencing respiratory illness then cultured in HEp-2 cells. We constructed plasmids expressing shRNAs. Tat peptide as a facilitator for shRNA plasmid delivery was used. The cytotoxicity of ribavirin, shRNA constructs, and control agents was assessed using the MTT assay. The transfection efficiency of Tat peptide-mediated shRNA delivery with that of lipofectamine 3000TM were compared. Finally, real-time PCR was employed to quantify HRSV replication in the treated cells. Tat peptide-mediated delivery of shRNA plasmids significantly suppressed the expression of the M and F genes of HRSV compared to lipofectamine 3000TM. This suppression was evident in both short-term experiments and scenarios involving stable shRNA expression. Furthermore, the combination of ribavirin with shRNA treatment resulted in a substantial reduction in viral load. Notably, the most pronounced antiviral effect was observed when both shRNAs were employed simultaneously. Our findings suggest that Tat peptide-mediated delivery of shRNA plasmids holds significant potential for achieving stable suppression of HRSV genes. This approach warrants further investigation as a potential gene therapy strategy for HRSV. By demonstrating promising results in vitro, this study highlights the need for future in vivo studies to comprehensively evaluate the therapeutic potential of this approach in a clinical setting.

Acid-Activated TAT Peptide-Modified Biomimetic Boron Nitride Nanoparticles for Enhanced Targeted Codelivery of Doxorubicin and Indocyanine Green: A Synergistic Cancer Photothermal and Chemotherapeutic Approach.

The evolution of nano-drug delivery systems addresses the limitations of conventional cancer treatments with stimulus-responsive nanomaterial-based delivery systems presenting temporal and spatial advantages. Among various nanomaterials, boron nitride nanoparticles (BNNs) demonstrate significant potential in drug delivery and cancer treatment, providing a high drug loading capacity, multifunctionality, and low toxicity. However, the challenge lies in augmenting nanomaterial accumulation exclusively within tumors while preserving healthy tissues. To address this, we introduce a novel approach involving cancer cell membrane-functionalized BNNs (CM-BIDdT) for the codelivery of doxorubicin (Dox) and indocyanine green to treat homologous tumor. The cancer cell membrane biomimetic CM-BIDdT nanoparticles possess highly efficient homologous targeting capabilities toward tumor cells. The surface modification with acylated TAT peptides (dTAT) further enhances the nanoparticle intracellular accumulation. Consequently, CM-BIDdT nanoparticles, responsive to the acidic tumor microenvironment, hydrolyze amide bonds, activate the transmembrane penetrating function, and achieve precise targeting with substantial accumulation at the tumor site. Additionally, the photothermal effect of CM-BIDdT under laser irradiation not only kills cells through thermal ablation but also destroys the membrane on the surface of the nanoparticles, facilitating Dox release. Therefore, the fabricated CM-BIDdT nanoparticles orchestrate chemo-photothermal combination therapy and effectively inhibit tumor growth with minimal adverse effects, holding promise as a new modality for synergistic cancer treatment.

Membrane-Active All-Hydrocarbon-Stapled α-Helical Amphiphilic Tat Peptides: Broad-Spectrum Antibacterial Activity and Low Incidence of Drug Resistance.

Multidrug resistance against conventional antibiotics has dramatically increased the difficulty of treatment and accelerated the need for novel antibacterial agents. The peptide Tat (47-57) is derived from the transactivating transcriptional activator of human immunodeficiency virus 1, which is well-known as a cell-penetrating peptide in mammalian cells. However, it is also reported that the Tat peptide (47-57) has antifungal activity. In this study, a series of membrane-active hydrocarbon-stapled α-helical amphiphilic peptides were synthesized and evaluated as antibacterial agents against Gram-positive and Gram-negative bacteria, including multidrug-resistant strains. The impact of hydrocarbon staple, the position of aromatic amino acid residue in the hydrophobic face, the various types of aromatic amino acids, and the hydrophobicity on bioactivity were also investigated and discussed in this study. Among those synthesized peptides, analogues P3 and P10 bearing a l-2-naphthylalanine (Φ) residue at the first position and a Tyr residue at the eighth position demonstrated the highest antimicrobial activity and negligible hemolytic toxicity. Notably, P3 and P10 showed obviously enhanced antimicrobial activity against multidrug-resistant bacteria, low drug resistance, high cell selectivity, extended half-life in plasma, and excellent performance against biofilm. The antibacterial mechanisms of P3 and P10 were also preliminarily investigated in this effort. In conclusion, P3 and P10 are promising antimicrobial alternatives for the treatment of the antimicrobial-resistance crisis.

Rpt5-Derived Analogs Stimulate Human Proteasome Activity in Cells and Degrade Proteins Forming Toxic Aggregates in Age-Related Diseases.

Aging and age-related diseases are associated with a decline in the capacity of protein turnover. Intrinsically disordered proteins, as well as proteins misfolded and oxidatively damaged, prone to aggregation, are preferentially digested by the ubiquitin-independent proteasome system (UIPS), a major component of which is the 20S proteasome. Therefore, boosting 20S activity constitutes a promising strategy to counteract a decrease in total proteasome activity during aging. One way to enhance the proteolytic removal of unwanted proteins appears to be the use of peptide-based activators of the 20S. In this study, we synthesized a series of peptides and peptidomimetics based on the C-terminus of the Rpt5 subunit of the 19S regulatory particle. Some of them efficiently stimulated human 20S proteasome activity. The attachment of the cell-penetrating peptide TAT allowed them to penetrate the cell membrane and stimulate proteasome activity in HEK293T cells, which was demonstrated using a cell-permeable substrate of the proteasome, TAS3. Furthermore, the best activator enhanced the degradation of aggregation-prone α-synuclein and Tau-441. The obtained compounds may therefore have the potential to compensate for the unbalanced proteostasis found in aging and age-related diseases.

Construction of a versatile fusion protein for targeted therapy and immunotherapy.

Antibody (Ab)-based drugs have been widely used in targeted therapies and immunotherapies, leading to significant improvements in tumor therapy. However, the failure of Ab therapy due to the loss of target antigens or Ab modifications that affect its function limits its application. In this study, we expanded the application of antibodies (Abs) by constructing a fusion protein as a versatile tool for Ab-based target cell detection, delivery, and therapy. We first constructed a SpaC Catcher (SpaCC for short) fusion protein that included the C domains of Staphylococcal protein A (SpaC) and the SpyCatcher. SpaCC conjugated with SpyTag-X (S-X) to form the SpaCC-S-X complex, which binds non-covalently to an Ab to form the Ab-SpaCC-S-X protein complex. The "X" can be a variety of small molecules such as fluoresceins, cell-penetrating peptide TAT, Monomethyl auristatin E (MMAE), and DNA. We found that Ab-SpaCC-S-FITC(-TAT) could be used for target cell detection and delivery. Besides, we synthesized the Ab-SpaCC-SN3-MMAE complex by linking Ab with MMAE by SpaCC, which improved the cytotoxicity of small molecule toxins. Moreover, we constructed an Ab-DNA complex by conjugating SpaCC with the aptamer (Ap) and found that Ab-SpaCC-SN3-Ap boosted the tumor-killing function of T-cells by retargeting tumor cells. Thus, we developed a multifunctional tool that could be used for targeted therapies and immunotherapies, providing a cheap and convenient novel drug development strategy.

Targeted degradation of hexokinase 2 for anti‑inflammatory treatment in acute lung injury.

Acute lung injury (ALI) is an acute inflammatory lung disease associated with both innate and adaptive immune responses. Hexokinase 2 (HK2) is specifically highly expressed in numerous types of inflammation‑related diseases and models. In the present study in vitro and in vivo effects of targeted degradation of HK2 on ALI were explored. The degradation of HK2 by the targeting peptide TAT (transactivator of transcription protein of HIV‑1)‑ataxin 1 (ATXN1)‑chaperone‑mediated autophagy‑targeting motif (CTM) was demonstrated by ELISA and western blotting in vitro and in vivo. The inhibitory effects of TAT‑ATXN1‑CTM on lipopolysaccharide (LPS)‑induced inflammatory responses were examined using ELISAs. The therapeutic effects of TAT‑ATXN1‑CTM on LPS‑induced ALI were examined via histological examination and ELISAs in mice. 10 µM TAT‑ATXN1‑CTM administration decreased HK2 protein expression and the secretion of proinflammatory cytokines (TNF‑α and IL‑1β) without altering HK2 mRNA expression in LPS‑treated both in vitro and in vivo, while pathological lung tissue damage and the accumulation of leukocytes, neutrophils, macrophages and lymphocytes in ALI were also significantly suppressed by 10 µM TAT‑ATXN1‑CTM treatment. TAT‑ATXN1‑CTM exhibited anti‑inflammatory activity in vitro and decreased the severity of ALI in vivo. HK2 degradation may represent a novel therapeutic approach for ALI.

Up-to-date Combinational Polymeric Approaches for the Enhancement of Drug Absorption and Membrane Transductivity

Abstract: Many of the challenges concerning pharmaceutical compounds are involved in the tuning of their physical properties and controlled and targeted drug delivery, in order to acquire the optimum therapeutic effect. With these challenges in mind and with the recent emergence of advanced materials, various combinational polymeric approaches have been applied. The use of coordination polymers, which are hybrid inorganic-organic materials, consists of transition metals and multidentate organic ligands and have been proven to prolong the release and increase the drug permeation rate of active pharmaceutical ingredients (APIs). Another recent approach is the preparation of deformable polymeric nanoparticles (DPN) that require the physical incorporation of a lipid in a polymeric micelle, offering flexible and deformable phase properties. It has been shown that skin delivery efficiency could be increased due to this deformable phase. Enhanced skin permeation was also observed when TAT peptides were chemically attached to the DPNs. Other recent approaches, such as microarray patches, sustained release microspheres, nanoparticles coated with biological membranes, nanosponges, and lipid-based nanoparticles, modified by polymers, have also been able to improve the pharmacokinetic profiles of APIs and are also discussed in this paper.