title: Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking.
abstract: Cyclotides or cyclic cystine-knot peptides have emerged as a promising class of pharmacological ligands that modulate protein function. Interestingly, very few cyclotides have been shown to enter into cells. Yet, it remains unknown whether backbone cyclization is required for their cellular internalization. In this report, we studied the cellular behavior of EETI-II, a model acyclic cystine-knot peptide. Even though synthetic methods have been used to generate EETI-II, recombinant methods that allow efficient large scale biosynthesis of EETI-II have been lagging. Here, we describe a novel protocol for recombinant generation of folded EETI-II in high yields and to near homogeneity. We also uncover that EETI-II is efficiently uptaken via an active endocytic pathway to early endosomes in mammalian cells, eventually accumulating in late endosomes and lysosomes. Notably, co-incubation with a cell-penetrating peptide enhanced the cellular uptake and altered the trafficking of EETI-II, leading to its evasion of lysosomes. Our results demonstrate the feasibility of modulating the subcellular distribution and intracellular targeting of cystine-knot peptides, and hence enable future exploration of their utility in drug discovery and delivery.
authors.affiliation: Department of Early Discovery Biochemistry, Genentech, South San Francisco, California.
authors.forename: Xinxin
authors.initials: X
authors.lastname: Gao
authors.affiliation: Department of Early Discovery Biochemistry, Genentech, South San Francisco, California.
authors.forename: Karen
authors.initials: K
authors.lastname: Stanger
authors.affiliation: Department of Early Discovery Biochemistry, Genentech, South San Francisco, California.
authors.forename: Harini
authors.initials: H
authors.lastname: Kaluarachchi
authors.affiliation: Department of Structural Biology, Genentech, South San Francisco, California.
authors.forename: Till
authors.initials: T
authors.lastname: Maurer
authors.affiliation: Department of Early Discovery Biochemistry, Genentech, South San Francisco, California.
authors.forename: Paulina
authors.initials: P
authors.lastname: Ciepla
authors.affiliation: Department of Pathology, Genentech, South San Francisco, California.
authors.forename: Cecile
authors.initials: C
authors.lastname: Chalouni
authors.affiliation: Department of Structural Biology, Genentech, South San Francisco, California.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Franke
authors.affiliation: Department of Early Discovery Biochemistry, Genentech, South San Francisco, California.
authors.forename: Rami N
authors.initials: RN
authors.lastname: Hannoush
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D057846:Cell-Penetrating Peptides / Q000378:metabolism; D003500:Cyclization / Q000502:physiology; D047169:Cyclotides / Q000378:metabolism; D003553:Cystine / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D010456:Peptides, Cyclic / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology*
chemical_list: D057846:Cell-Penetrating Peptides; D047169:Cyclotides; D010456:Peptides, Cyclic; D010940:Plant Proteins; C063950:trypsin inhibitor EETI II protein, plant; D003553:Cystine