Part:BBa_K3090000
cell penetrating peptide
Nuclear localization signal (NLS) enriched in positively charged residues that can function as a Cell-penetrating peptide (CPP), similar to the classical CPP derived from HIV type 1 transactivator of transcription protein (HIV TAT).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
This part(BBa_K3090000) was fused to the N-terminus of scFv(P5) (part number BBa_K3090001) to design a cell-penetrating antibody fragment (part BBa_K3090002). The sequence of the designed protein is as follows.
MTYTRRRFRR RRHRPRS QVQLQESGGD LVQPGGSLKL SCAVSGFSLT GYGVNWVRQT PDKRLEWVAM IWGDGNTDYN SSVKGRFTIS KDNAKSTVYL QMSSLKSEDT AMYYCARERD YRLDYWGQGT TVTVSS GGGGSGGGGSGGGGS DIELTQSPAS LAVSLGQRAT ISCRASGNIH NYLAWYQQKP GQPPKLLIYY TTTLADGIPA RFSGSGSGTD YTLTINPVEA DDVATYYCQH FWSTPRTFGG GTKLEIKR
We modified the buforin IIb peptide sequence to induce cancer cell death by delivering doxorubicin and siRNA. Herein, CPP buforin IIb, a synthetic analog of antimicrobial peptide buforin II, presented cytotoxicity at high concentrations: cytotoxicity and alpha-helicity are understood to be directly related, and CPP interaction with gangliosides, phosphatidylserine, and heparan sulfate inducing buforin IIb internalization upregulates caspase-9 activation and cytosolic cytochrome c release in situ. Hence, modification of buforin IIb with a motif of stepwise elimination C-terminal (-RLLR) for the reduction of alpha-helicity was considered in our project, presented below.
Modify buforin IIb peptide - amino acid sequence modification
Buforin IIB: RAGLQFPVGRLLRRLLRRLLR (21 aa) (charge = +7)
Modified Version 1 (MV1): RAGLQFPVGRLLRRLLR (17 aa) (charge = +5)
Modified Version 2 (MV2): RAGLQFPVGRLLR (13 aa) (charge = +3)
Further modification: FITC was added to the c-terminal of the peptide to analyze the intracellular distribution in cancer cells. Doxorubicin was added to the c-terminal of the peptide to analyze the drug sensitivity of doxorubicin (chemotherapy drug).
Experimentally, colorimetric MTT metabolic (cytotoxicity) assay validated cell viability of ~40 mM MV1 and MV2. FITC confocal microscopy presented the highest internalization and transfection efficacy at 40 mM MV1. RT-PCR assay reported the lowest band intensity quantification at CYP1A1 siRNA (20µM) + 40 mM MV1 (0.2 pdu, normalized). We believe the integration of both literature and results can be compatible for teams devising synthetic construct to cell line-specific drug delivery via modification of affinity peptide and conjugation of sensitivity-inducing agents.
CPPs are positively charged short peptides with 5–30 amino acids that can penetrate biological membranes and deliver various cargos into cells. CPPs have received extensive attention in recent decades due to their high transduction efficiency and low cytotoxicity. Furthermore, owing to the ability of these peptide sequences to transport across the cellular membrane, they were found to be a promising candidate for intracellular delivery.
Two methods are available for conjugation of CPP to cargo molecules: covalent and non-covalent binding. In covalent conjugation, cargo molecules attach to CPP via a covalent bond in a time-consuming process. However, this method has a drawback when transporting various cargo because each type of cargo needs its covalent conjugation. In the second approach, the electrostatic interaction of CPPs binds to cargo. Due to its high flexibility, this method is suitable for a wide range of cargo delivery applications. Overall, CPPs can apply for diagnostic and therapeutic applications, such as the delivery of fluorescent compounds for imaging, transportation of peptides and proteins for cancer therapy, and penetration of molecules into induced pluripotent stem cells for directing differentiation.
In 2019, a paper is published about the efficiency of fusion peptides for intracellular delivery of therapeutic antibodies.
<Reference> Gaston, J., Maestrali, N., Lalle, G., Gagnaire, M., Masiero, A., Dumas, B., Dabdoubi, T., Radošević, K., Berne, P.-F. Intracellular delivery of therapeutic antibodies into specific cells using antibody-peptide fusions (2019) Scientific Reports, 9 (1), art. no. 18688,
In this paper, the authors investigated various fusion peptides for their efficiency.
Name CPP_length Properties Net_charge CPP_Sequence
Pep-1 21 Amphipathic 3 KETWWETWWTEWSQPKKKRKV
TAT 11 Cationic 8 YGRKKRRQRRR
PEPth 12 Cationic 5 VKKKKIKAEIKI
aurein 1.2 13 Amphipathic 1 GLFDIIKKIAESF
MTS 17 Hydrophobic 0 KGEGAAVLLPVLLAAPG
GFWFG 5 Hydrophobic 0 GFWFG 31
Above peptides are attached to different positions of antibody (N-terminal of light or heavy chain, C-terminal of light or heavy chain, hinge region of heavy chain) and the antibody yield and efficiency of delivery were investigated. The result showed Pep-1 and PEPth were best when attached to C-terminal of light chain or hinge region.
We believe this study can be useful for teans who wants to design a fusion peptide to deliver an anitbody into the cell.
References
1: Yu W, Zhan Y, Xue B, Dong Y, Wang Y, Jiang P, Wang A, Sun Y, Yang Y. Highly efficient cellular uptake of a cell-penetrating peptide (CPP) derived from the capsid protein of porcine circovirus type 2. J Biol Chem. 2018 Sep 28;293(39):15221-15232.
2. Gaston, J., Maestrali, N., Lalle, G., Gagnaire, M., Masiero, A., Dumas, B., Dabdoubi, T., Radošević, K., Berne, P.-F. Intracellular delivery of therapeutic antibodies into specific cells using antibody-peptide fusions (2019) Scientific Reports, 9 (1), art. no. 18688
3. Lee HS, Park CB, Kim JM, Jang SA, Park IY, Kim MS, Cho JH, Kim SC. Mechanism of anticancer activity of buforin IIb, a histone H2A-derived peptide. Cancer Lett. 2008 Nov 18;271(1):47-55. doi: 10.1016/j.canlet.2008.05.041. Epub 2008 Jul 9. PMID: 18617323.
1. A. Prochiantz et al. Messenger proteins: homeoproteins, TAT and others Curr. Opin. Cell Biol., 12 (4) (2000), pp. 400-406
2. J.-H. Kang, M.-Y. Jung, X. Yin, M. Andrianifahanana, D.M. Hernandez, E.B. Leof Cell-penetrating peptides selectively targeting SMAD3 inhibit profibrotic TGF-β signaling J. Clin. Invest., 127 (7) (2017), pp. 2541-2554
3. K. Kurrikoff, M. Gestin, Ü. Langel Recent in vivo advances in cell-penetrating peptide-assisted drug delivery Expert Opin. Drug Deliv., 13 (3) (2016), pp. 373-387
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