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Novel cell-penetrating peptide targeting mitochondria
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-7769-6905
Stockholm University, Faculty of Science, Department of Neurochemistry. University of Sassari, Italy.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-6107-0844
2015 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 29, no 11, p. 4589-4599Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) are short, nontoxic peptides with cationic and/or amphipathic properties able to cross the cellular membrane. CPPs are used for the delivery of a wide variety of cargoes, such as proteins, oligonucleotides, and therapeutic molecules. The aim of the present study was to synthesize unusually small novel CPPs targeting mitochondria based on the Szeto-Schiller peptide (SS-31) to influence intramitochondrial processes and to improve the biologic effects. All the peptides used were synthesized manually using 9-fluorenylmethyloxycarbonyl chemistry. In the first part of the study, HeLa 705, U87, and bEnd.3 cells were used as in vitro delivery model. Cells were incubated for 24 h at 37°C and 5% CO2 with different concentrations of our peptides. Cell proliferation assay was performed to evaluate cell viability. Biologic effects such as mitochondrial membrane potential and antioxidant activity were evaluated. H2O2 was used as positive control. Uptake studies were performed using peptides conjugated with 5(6)-carboxyfluorescein (FAM). Fluorescent microscopy was used to determine presence and localization of peptides into the cells. Isolated mitochondria from pretreated cells and mitochondria treated after isolation were used to confirm the targeting ability of the peptide. Uptake of FAM alone was used as negative control. Microscopy studies confirmed the ability of peptides to penetrate cell. Localization analysis showed increase in uptake by 35% compared with SS-31. Mitochondrial CPP 1 (mtCPP-1) had no effect on mitochondrial membrane potential and prevented reactive oxygen species formation in bEnd.3 cells by 2-fold compared with SS-31. No cytotoxicity was observed even at high concentration (100 µM). These data suggest that mtCPP-1 is a mitochondrial CPP and protect mitochondria from oxidative damage due to its own antioxidant activities.-Cerrato, C. P., Pirisinu M., Vlachos E. N., Langel, Ü. Novel cell-penetrating peptide targeting mitochondria.

Place, publisher, year, edition, pages
2015. Vol. 29, no 11, p. 4589-4599
Keywords [en]
mitochondrial targeting, ROS reduction, mitochondrial membrane potential, antioxidant activity
National Category
Biological Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-122077DOI: 10.1096/fj.14-269225ISI: 000364514700018PubMedID: 26195590OAI: oai:DiVA.org:su-122077DiVA, id: diva2:862760
Available from: 2015-10-23 Created: 2015-10-23 Last updated: 2018-04-16Bibliographically approved
In thesis
1. Cell-penetrating peptide targeting mitochondria: Design, synthesis, characterization, and biological effects
Open this publication in new window or tab >>Cell-penetrating peptide targeting mitochondria: Design, synthesis, characterization, and biological effects
2015 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

More than twenty years after the discovery of the first cell-penetrating peptide (CPP), a large number of both naturally occurring as well as engineered CPPs have been discovered. Generally, CPPs are short polycationic sequences of less than 30 amino acids that are able to translocate different cargoes into cells. They are amphipathic and net positively charged at physiological pH. The cargo can be covalently attached to the CPP, which can be achieved by expression as a fusion construct or by chemical coupling; or the cargo and carrier could bind each other non-covalently mainly through ionic interactions.

A series of CPPs targeting mitochondria (mtCPPs) were studied in an effort to optimize their applications for the reduction of reactive oxygen species targeting this therapeutically important organelle. Mitochondria have evolved to play a vital role in both life and death of eukaryotic cells, through involvement in numerous cellular functions, such as the proficient production of energy from ATP biosynthesis and the regulation of programmed cell death. As a result, dysfunction in the biochemical processes housed within this organelle is implicated in diverse diseases, including cancer, diabetes, and neurodegenerative disorders. Advancing mitochondrial medicine by probing the subcellular biochemistry or targeting therapeutics into this organelle has motivated the development of effective mitochondrial delivery vectors. A fluorescent probe was covalently attached at the N-terminus of the analog peptides to determine the cellular internalization and the possibility to be transported to mitochondria by mtCPPs. The results report the development of a novel cationic peptides (mtCPP-1), which is readily cell permeable and preferentially localize into the mitochondria of living mammalian cells. By substitutions with both natural and synthetic amino acids, and monitoring the intracellular localization by fluorescence microscopy, the mitochondrial accumulation with a cationic peptide was achieved. The biological and chemical characterization of mtCPP-1 revealed the importance of balancing the opposing characteristics of positive charge and lipophilicity to attain preferential sequestration into mitochondria, as well as provide evidence that this antioxidant peptide will be suitable as mitochondrial delivery vector.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, 2015. p. 47
Keywords
cell-penetrating peptide, mitochondrial targeting, ROS reduction, mitochondrial membrane potential, antioxidant activity
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-122082 (URN)978-91-7649-312-0 (ISBN)
Presentation
2015-11-30, Heilbronsalen, C458, Svante Arrhenius väg 16B, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2015-11-17 Created: 2015-10-23 Last updated: 2015-11-17Bibliographically approved
2. Cell-Penetrating Peptides for Mitochondrial Targeting
Open this publication in new window or tab >>Cell-Penetrating Peptides for Mitochondrial Targeting
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mitochondria have simply been known as the cell’s powerhouse for a long time, with its vital function of producing ATP. However, substantially more attention was directed towards these organelles once they were recognized to perform several essential functions having an impact in cell biology, pharmaceutics and medicine. Dysfunctions of these organelles have been linked to several diseases such as diabetes, cancer, neurodegenerative diseases and cardiovascular disorders. Mitochondrial medicine emerged once the relationship of reactive oxygen species and mutations of the mitochondrial DNA linked to diseases was shown, referred to as mitochondrial dysfunction. This has led to the need to deliver therapeutic molecules in their active form not only to the target cells but more importantly into the targeted organelles.

In this thesis, cell-penetrating peptides (CPPs) used as mitochondrial drug delivery system and the pathways involved in the uptake mechanisms of a CPP are described. In particular, Paper I describes a novel cell-penetrating peptide targeting mitochondria with intrinsic antioxidant properties. Paper II expands upon this first finding and show that the same peptide can carry a glutathione analogue peptide with improved radical scavenging ability into cytoplasm and mitochondria. Paper III introduces mitochondrial targeting peptides for delivery of therapeutic biomolecules to modify mitochondrial gene expression. In Paper IV, the uptake mechanisms of the CPP delivery strategy has been investigated to gain a better understanding of the used transfection system.

Overall, this thesis summarizes our current effort regarding cell-penetrating peptides delivery system to target mitochondria and the progress made towards a potential gene therapy. It contributes to the field of CPPs and drug delivery with a set of peptides with radical scavenging ability, a strategy to deliver oligonucleotides to mitochondria as proof-of-concept for mitochondrial gene therapy, and to help understanding the pathways involved in CPPs uptake.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018. p. 62
Keywords
Mitochondrial targeting, cell-penetrating peptides, antioxidant activity, scavenging ability, oligonucleotide delivery
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-155156 (URN)978-91-7797-230-3 (ISBN)978-91-7797-231-0 (ISBN)
Public defence
2018-06-01, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Available from: 2018-05-08 Created: 2018-04-13 Last updated: 2018-05-04Bibliographically approved

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