A comprehensive in silico characterization of bacterial signal peptides for the excretory production of Anabaena variabilis phenylalanine ammonia lyase in Escherichia coli

Hemmati

2019

Molecules

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.

Section: Resultsmentioning

confidence: 73%

Section: Resultsmentioning

confidence: 99%

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

Hemmati

2019

Molecules

“…The GRAVY value for a peptide is defined by adding the hydropathy index of each residue divided by the sequence length. A more negative value indicates that the peptide is more hydrophilic ( Owji and Hemmati, 2018 ). GRAVY values for SCV2s-CPPs range from −3.3 to 2.2.…”

Section: Resultsmentioning

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

Infection, Genetics and Evolution

Haddad

2020

Self Cite

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.

“…To stretch the boundary of melanogenesis inhibitors, an enzyme such as TAL deviates the substrate needed for melanin biosynthesis. The other member of ammonia‐lyases named phenylalanine ammonia‐lyase (PAL) has been used to produce a recombinant medication called Palynziq for phenylketonuric patients 33 . Hence, TAL can be a promising treatment for L‐Tyr‐related disorders.…”

Section: Discussionmentioning

confidence: 99%

“…The other member of ammonia-lyases named phenylalanine ammonia-lyase (PAL) has been used to produce a recombinant medication called Palynziq for phenylketonuric patients. 33 Hence, TAL can be a promising treatment for L-Tyr-related disorders. Since the enzyme cannot pass through the cell membrane, we could achieve this goal by using the advantages of CPPs in cova- 34 Therefore, in the current study, a TAT-RsTAL fusion protein was generated, and its potential for decreasing the tyrosine content in the intracellular compartment, and consequently, the inhibition of melanogenesis in B16F10 cells was examined.…”

Section: Further In Silico Evaluation Of Tat-rstal Constructmentioning

confidence: 99%

Introducing a delivery system for melanogenesis inhibition in melanoma B16F10 cells mediated by the conjugation of tyrosine ammonia‐lyase and a TAT‐penetrating peptide

Sadeghian

Bahraman

et al. 2020

Biotechnology Progress

Self Cite

Hyperpigmentation disorders negatively influence an individual's quality of life and may cause emotional distress. Over the years, various melanogenesis inhibitors (mainly tyrosinase inhibitors) have been developed, most of which with low efficacy or high toxicity. Although metabolic engineering by deviation in the flux of substrate is of considerable interest, trials to develop a melanogenesis inhibitor based on L-tyrosine (L-Tyr) restriction are missing. We propose a novel proteinaceous melanogenesis inhibitor called tyrosine ammonia-lyase (TAL), an enzyme that catalyzes the conversion of L-Tyr to p-coumaric acid and ammonia. Since the cell membrane can act as a barrier for intracellular protein delivery, we have covalently conjugated a recombinant TAL enzyme from Rhodobacter sphaeroides (RsTAL) to a trans-activator of transcription (TAT) cell-penetrating peptide (CPP) to afford the intracellular delivery. The heterologously expressed TAT-RsTAL fusion protein was delivered successfully into B16F10 melanocytes as confirmed by the direct fluorescence microscopy with increased intensity from 30 to 180 min. TAT-RsTAL showed sufficient intracellular activity of about 0.83 ± 0.04 and 0.34 ± 0.03 nmol•mg −1 •s −1 for the native and inclusion body-extracted conjugates, respectively. The conjugate inhibited melanin biosynthesis in B16F10 cells in a time-dependent manner. Melanin accumulation was inhibited by 12.7 ± 6.2%, 28.2 ± 5.7%, and 33.9 ± 2.9% compared to the nontreated control groups after 24, 48, and 72 hr of incubation, respectively. L-Tyr restriction had no significant effect on the cell viability up to a concentration of 100 μgml −1 even after 72 hr. According to the observed hypopigmentary effect of the conjugate in this study, TAT-RsTAL can be suggested as a melanogenesis inhibitor for further investigations.