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Cellular translocation of proteins by transportan
Stockholms universitet / Estonian Biocentre, Tartu, Estonia.
Södertörn University, Avdelning Naturvetenskap. Stockholms universitet.
Södertörn University, Avdelning Naturvetenskap. Stockholms universitet.
Stockholms universitet.
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2001 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 15, no 8, 1451- p.Article in journal (Refereed) Published
Abstract [en]

Proteins with molecular masses ranging from 30 kDa. (green fluorescent protein, GFP) to 150 kDa (monoclonal and polyclonal antibodies) were coupled to the cellular translocating peptide transportan. We studied the ability of the resulting protein-peptide constructs to penetrate into Bowes melanoma, BRL, and COS-7 cells. After 0.5-3 h incubation with recombinant GFP coupled to transportan, most of the GFP fluorescence was found in intracellular membranes of BRL and COS-7 cells, which suggests that transportan could internalize covalently linked proteins of about 30 kDa in a folded state. Transportan could internalize covalently coupled molecules of even larger size; that is, avidin and antibodies, (up to 150 kDa). The covalent bond between the transport peptide and its cargo is not obligatory because streptavidin was translocated into the cells within 15 min as a noncovalent complex with biotinylated transportan. Inside the cells, the delivered streptavidin was first located mainly in close proximity to the plasma membrane and was later distributed to the perinuclear region. Most of the internalized streptavidin was confined to vesicular structures, but a significant fraction of the protein was distributed in the cytoplasm. Our data suggest that transportan can deliver proteins and other hydrophilic macromolecules into intact mammalian cells, and this finding demonstrates good potential as powerful cellular delivery vector for scientific and therapeutic purposes.

Place, publisher, year, edition, pages
2001. Vol. 15, no 8, 1451- p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:sh:diva-15859DOI: 10.1096/fj.00-0780fjeISI: 000173705800015OAI: oai:DiVA.org:sh-15859DiVA: diva2:509230
Available from: 2012-03-12 Created: 2012-03-09 Last updated: 2017-02-13Bibliographically approved
In thesis
1. Targeting of a nascent integral membrane protein to the nuclear pores and its degradation during apoptosis
Open this publication in new window or tab >>Targeting of a nascent integral membrane protein to the nuclear pores and its degradation during apoptosis
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Place, publisher, year, edition, pages
Stockholm: Stockholms universitet, 2002. 46 p.
National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-31521 (URN)91-7265-559-3 (ISBN)
Public defence
2002-12-13, Magnélisalen, Kemiska Övningslaboratoriet, Svante Arrenihusväg 12, Stockholm, 10:00
Opponent
Supervisors
Available from: 2016-12-22 Created: 2016-12-22 Last updated: 2016-12-22Bibliographically approved
2. Dynamic aspects of nucleocytoplasmic trafficking
Open this publication in new window or tab >>Dynamic aspects of nucleocytoplasmic trafficking
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cellular structures and compartmentalization is the result of a dynamic steady state exchange between its components. This thesis is focused in investigations of dynamic properties of green fluorescent protein (GFP)-labeled proteins in live cells using confocal laser microscopy in combination with bleaching techniques such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP).

Studies of dynamic properties of c-Myc in living cells showed that c-Myc is shuttling between the nucleus and the cytoplasm. c-Myc also enters the nucleoli during certain conditions. Nucleolar c-Myc is dynamically associated to structural component(s) of nucleoli, but can exchange with soluble pools in the nucleoplasm and cytoplasm.

Photobleaching experiments showed that a significant fraction of HIV-1 Vpr is dynamically associated with the NE and rapidly exchanges between the nucleoplasm and the cytoplasm. The yeast two-hybrid system, pull-down experiments and co-immunoprecipitating was used to show that Vpr interacts specifically and directly with a domain in the N-terminal portion of the NPC protein hCG1. The results suggest that the specific interaction of HIV-1 Vpr with the nucleoporin hCG1 results in the dynamic retention of Vpr at the nuclear envelope.

The distribution and dynamic properties of NPC proteins was investigated in NIH/3T3 cells, lacking the pore membrane protein gp210. Confocal laser scanning microscopy and FRAP experiments showed that the absence of gp210 from nuclear pores of NIH/3T3 cells did neither alter the distribution nor dynamic properties of POM121 and NUP107 (two NPC proteins stably integrated in the NPC).

Degradation of the integral nuclear pore membrane protein POM121 during apoptosis was investigated in relation to other apoptotic events. POM121 cleavage, which is the earliest sign of dismantling of the nuclear membrane, is due to caspase-3-dependent cleavage at aspartate-531. Loss of nuclear compartmentalization in live cells undergoing apoptosis was monitored as appearance of GFP-NLS in the cytoplasm. The time of appearance of cytoplasmic GFP-NLS correlated with caspase-3-dependent cleavage of POM121. Both events occured concomitantly with collapsing of chromatin against the nuclear periphery, but preceded the onset of nucleosomal DNA fragmentation.

Translocation ability of the cell-penetrating peptide, transportan, into living cells was investigated. Recombinantly expressed GFP was purified and conjugated to chemically synthesized transportan via a disulfide bond and added to tissues culture cells. Transportan was able to internalize a 27 kDa protein such as GFP in a native folded state into living cells.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, Stockholms universitet, 2004. 41 p.
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:sh:diva-32045 (URN)91-7265-803-7 (ISBN)
Public defence
2004-05-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2017-02-13 Created: 2017-02-13 Last updated: 2017-02-13Bibliographically approved

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