Identification of proteins released by mammalian cells that mediate DNA internalization through proteoglycan-dependent macropinocytosis

Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskrift

Abstract

Naked DNA plasmid represents the simplest vehicle for gene therapy and DNA-based vaccination purposes; however, the, molecular mechanisms of DNA uptake in mammalian cells are poorly understood. Here, we show that naked DNA uptake occurs via proteoglycan-dependent macropinocytosis, thus challenging the concept of a specific DNA-internalizing receptor. Cells genetically deficient in proteoglycans, which constitute a major source of cell-surface polyanions, exhibited substantially decreased uptake of likewise polyanionic DNA. The apparent paradox was explained by the action of DNA-transporting proteins present in conditioned medium. Complexes between these proteins and DNA require proteoglycans for cellular entry. Mass spectrometry analysis of cell medium components identified several proteins previously shown to associate with DNA and to participate in membrane transport of macromolecular cargo. The major pathway for proteoglycan-dependent DNA uptake was macropinocytosis, whereas caveolae-dependent and clathrin-dependent pathways were not involved, as determined by using caveolin-1 knock-out cells, dominant-negative constructs for dynamin and Eps15, and macropinocytosis- disruptive drugs, as well as confocal fluorescence co-localization studies. Importantly, a significant fraction of internalized DNA was translocated to the nucleus for expression. Our results provide novel insights into the mechanism of DNA uptake by mammalian cells and extend the emerging role of proteoglycans in macromolecular transport.

Detaljer

Författare
Enheter & grupper
Forskningsområden

Ämnesklassifikation (UKÄ) – OBLIGATORISK

  • Infektionsmedicin
  • Cancer och onkologi

Nyckelord

Originalspråkengelska
Sidor (från-till)27897-27904
Antal sidor8
TidskriftJournal of Biological Chemistry
Volym282
Utgåva nummer38
StatusPublished - 2007 sep 21
PublikationskategoriForskning
Peer review utfördJa

Relaterad forskningsoutput

Anders Wittrup, 2009, Section of Oncology, Dept of Clinical Sciences.. 130 s.

Forskningsoutput: AvhandlingDoktorsavhandling (sammanläggning)

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