TAT-mediated intracellular delivery of carboxypeptidase G2 protects against methotrexate-induced cell death in HepG2 cells

Sadeghian, Issa; Khalvati, Bahman; Ghasemi, Younes; Hemmati, Shiva

doi:10.1016/j.taap.2018.03.023

Cited by 25 publications

(15 citation statements)

References 54 publications

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“…In this study, “Tat (48–60)” and one of its derivatives “Rev (34–50)” are on our final candidate list. This is in agreement with our recent findings, wherein we have demonstrated experimentally the efficient intracellular delivery of CPG2 conjugated to the TAT peptide [28]. Both native and denatured forms of the enzyme transduced efficiently into the HepG2 cells in a concentration- and time-dependent manner.…”

Section: Resultssupporting

confidence: 93%

“…Although a bioinformatics method for the assessment of possible entrapment of CPP-protein conjugates is not available yet, using multivalent CPPs or CPP conjugation to a pH-Dependent Membrane Active Peptide (PMAP) are recommended to resolve inadequate CPP-protein endosomal release issues [79]. However, it was promising that in our experimental study conducted on the N-terminal conjugate of TAT-CPG2, the release of CPP-cargo conjugates from endosomes was high to display adequate pharmacological responses [28].…”

Section: Resultsmentioning

confidence: 99%

“…Accordingly, it seems reasonable to deliver the CPG2 enzyme to the intracellular compartment. Through experimental studies, we have recently achieved the intracellular delivery of CPG2 in conjugation with the well-known transcription transactivator protein of HIV-1 (TAT) peptide [28]. Therefore, CPG2 has been used as the model cargo in the preparation of the current investigation.…”

Section: Introductionmentioning

confidence: 99%

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Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule

Behzadipour

Hemmati

2019

Molecules

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Access of proteins to their intracellular targets is limited by a hydrophobic barrier called the cellular membrane. Conjugation with cell-penetrating peptides (CPPs) has been shown to improve protein transduction into the cells. This conjugation can be either covalent or non-covalent, each with its unique pros and cons. The CPP-protein covalent conjugation may result in undesirable structural and functional alterations in the target protein. Therefore, we propose a systematic approach to evaluate different CPPs for covalent conjugations. This guide is presented using the carboxypeptidase G2 (CPG2) enzyme as the target protein. Seventy CPPs —out of 1155— with the highest probability of uptake efficiency were selected. These peptides were then conjugated to the N- or C-terminus of CPG2. Translational efficacy of the conjugates, robustness and thermodynamic properties of the chimera, aggregation possibility, folding rate, backbone flexibility, and aspects of in vivo administration such as protease susceptibility were predicted. The effect of the position of conjugation was evaluated using unpaired t-test (p < 0.05). It was concluded that N-terminal conjugation resulted in higher quality constructs. Seventeen CPP-CPG2/CPG2-CPP constructs were identified as the most promising. Based on this study, the bioinformatics workflow that is presented may be universally applied to any CPP-protein conjugate design.

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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule

Behzadipour

Hemmati

2019

Molecules

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“…In studies using Hep3B hepatocellular carcinoma cells, toxicity was observed at 100 nM MTX [41]. In another study using another HepG2, toxicity was 43.7% when treated with 100 μM MTX for 24 h and 17.69% when treated for 48 h [42]. Hepatocyte spheroids were prepared by mixing THP-1 and hTERT-HSC cells in HepaRG in a hanging drop system; cell viability was below 50% in 30 μM MTX after a 14-day treatment [43].…”

Section: Discussionmentioning

confidence: 99%

Recapitulation of methotrexate hepatotoxicity with induced pluripotent stem cell-derived hepatocytes from patients with rheumatoid arthritis

Kim

Choi

et al. 2018

Stem Cell Res Ther

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BackgroundMethotrexate (MTX) is widely used for the treatment of rheumatoid arthritis (RA). The drug is cost-effective, but sometimes causes hepatotoxicity, requiring a physician’s attention. In this study, we simulated hepatotoxicity by treating hepatocytes derived from RA patient–derived induced pluripotent stem cells (RA-iPSCs) with MTX.MethodsRA-iPSCs and healthy control iPSCs (HC-iPSCs) were established successfully. RA-iPSCs were differentiated into hepatocytes in two-dimensional (2D) monolayers and three-dimensional (3D) hepatocyte spheroid cultures; this process required growth factors such as BMP4, bFGF, HGF, and OSM. Immunofluorescence staining and flow cytometry were performed to confirm that the mature hepatocytes expressed cytokeratin 18, anti–alpha-1 antitrypsin, and albumin. MTX toxicity was evaluated via monitoring of cell viability, alanine aminotransferase, and mitochondrial status after MTX treatment in 2D and 3D cultures.ResultsRA-iPSCs generated from three RA patients suffering from MTX-induced hepatotoxicity differentiated into the endoderm lineage, hepatoblasts, and hepatocytes. In 2D culture, RA-iPSC-derived hepatocytes were more sensitive to MTX than healthy controls. A 3D culture system using hepatocyte spheroids also successfully recapitulated MTX-induced hepatotoxicity. The 3D culture system had several advantages, including longer culture periods under more complex conditions.ConclusionsA patient-derived iPSC platform could recapitulate MTX toxicity. Simulation of drug toxicity in vitro may help clinicians choose safer drugs or less toxic doses.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-1100-1) contains supplementary material, which is available to authorized users.

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“…In the current study, finding and characterization of CPPs not only in novel insertions but also in the whole proteome of the virus was of paramount concern. To introduce the good out of the bad and the ugly, such CPPs could be mediators for potential drug delivery like the well-known TAT ( Sadeghian et al, 2018 ) or act as a bioactive peptide by themselves. This investigation might also improve our understanding of replication, transcription, and pathogenesis of the virus.…”

Section: Introductionmentioning

confidence: 99%

Decoding the proteome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for cell-penetrating peptides involved in pathogenesis or applicable as drug delivery vectors

Hemmati

Behzadipour

Haddad

2020

Infection, Genetics and Evolution

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Synthetic or natural derived cell-penetrating peptides (CPPs) are vastly investigated as tools for the intracellular delivery of membrane-impermeable molecules. As viruses are intracellular obligate parasites, viral originated CPPs have been considered as suitable intracellular shuttling vectors for cargo transportation. A total of 310 CPPs were identified in the proteome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Screening the proteome of the cause of COVID-19 reveals that SARS-CoV-2 CPPs (SCV2-CPPs) span the regions involved in replication, protein-nucleotide and protein-protein interaction, protein-metal ion interaction, and stabilization of homo/hetero-oligomers. However, to find the most appropriate peptides as drug delivery vectors, one might face several hurdles. Computational analyses showed that 94.3% of the identified SCV2-CPPs are non-toxins, and 38% are neither antigenic nor allergenic. Interestingly, 36.70% of SCV2-CPPs were resistant to all four groups of protease families. Nearly 1/3 of SCV2-CPPs had sufficient inherent or induced helix and sheet conformation leading to increased uptake efficiency. Heliquest lipid-binding discrimination factor revealed that 44.30% of the helical SCV2-CPPs are lipid-binding helices. Although Cys-rich derived CPPs of helicase (NSP13) can potentially fold into a cyclic conformation in endosomes with a higher rate of endosomal release, the most optimal SCV2-CPP candidates as vectors for drug delivery were SCV2-CPP118, SCV2-CPP119, SCV2-CPP122, and SCV2-CPP129 of NSP12 (RdRp). Ten experimentally validated viral-derived CPPs were also used as the positive control to check the scalability and reliability of our protocol in SCV2-CPP retrieval. Some peptides with a cell-penetration ability known as bioactive peptides are adopted as biotherapeutics themselves. Therefore, 59.60%, 29.63%, and 32.32% of SCV2-CPPs were identified as potential antibacterial, antiviral, and antifungals, respectively. While 63.64% of SCV2-CPPs had immuno-modulatory properties, 21.89% were recognized as anti-cancers. Conclusively, the workflow of this study provides a platform for profound screening of viral proteomes as a rich source of biotherapeutics or drug delivery carriers.

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TAT-mediated intracellular delivery of carboxypeptidase G2 protects against methotrexate-induced cell death in HepG2 cells

Cited by 25 publications

References 54 publications

Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule

Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule

Recapitulation of methotrexate hepatotoxicity with induced pluripotent stem cell-derived hepatocytes from patients with rheumatoid arthritis

Decoding the proteome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for cell-penetrating peptides involved in pathogenesis or applicable as drug delivery vectors

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