Difference between revisions of "Part:BBa K3090000"

Revision as of 12:44, 5 October 2021

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).

We modified buforin IIb peptide sequence to induce cancer cell death by delivering doxorubicin and siRNA.

Sequence and Features

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

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.

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.