Volume 11, Issue 2, Pages 205-214 (February 2005) A ubiquitin-based assay for the cytosolic uptake of protein transduction domains  Fabien Loison, Philippe Nizard, Tony Sourisseau, Pascale Le Goff, Laure Debure, Yves Le Drean, Denis Michel  Molecular Therapy  Volume 11, Issue 2, Pages 205-214 (February 2005) DOI: 10.1016/j.ymthe.2004.10.010 Copyright © 2004 Terms and Conditions

Fig. 1 Scheme of the construction of the PTD–Ub-linked proteins. (A) The cargo coding sequences were fused in frame as C-terminal extensions of ubiquitin by using a SfoI restriction site, whose upstream half-site corresponds to the carboxy-terminal glycine codon of ubiquitin. PTD coding sequences were inserted into a unique BamHI restriction site in front of ubiquitin. All proteins contain the amino-terminal region MRGSHHHHHH including a polyhistidine tag for affinity purification. (B) The five chimeric recombinant proteins used in the present study. Two PTD were tested: melittin (MEL) and the TAT peptide from HIV. Three cargo proteins were used: a 19-amino-acid-long peptide (PEP) and the Hsp70 and Bcl-XL proteins. If proteins reach the cytosolic compartment of target cells, the cargoes should be released by DUB processing. Molecular Therapy 2005 11, 205-214DOI: (10.1016/j.ymthe.2004.10.010) Copyright © 2004 Terms and Conditions

Fig. 2 Fluorometry analysis of cells incubated with fluorescent recombinant proteins containing no PTD (Ub-PEP), melittin (MEL-Ub-PEP), or the TAT peptide (TAT-Ub-PEP). As indicated in the scheme (A), cells were treated with trypsin (B) before or (C, D) after incubation with fluorescent proteins or free dye. (E) Comparison of cellular fluorescences obtained after 10-min- (white) and 12-h-long (black) incubations with proteins. Molecular Therapy 2005 11, 205-214DOI: (10.1016/j.ymthe.2004.10.010) Copyright © 2004 Terms and Conditions

Fig. 3 Microscopy analysis of transduction of TAT-Ub-PEP fusion proteins into cells. (A) MCF-7 cells were treated with either free Alexa Fluor 594 dye or Alexa Fluor 594-conjugated TAT-Ub-PEP proteins and then observed by fluorescence microscopy after organic fixation. (B) MCF-7 cells were incubated with high-activity TAT–β-galactosidase or β-galactosidase proteins and then washed and stained using the X-gal procedure. (C) Living cells incubated with fluorescent TAT-Ub-PEP proteins. Molecular Therapy 2005 11, 205-214DOI: (10.1016/j.ymthe.2004.10.010) Copyright © 2004 Terms and Conditions

Fig. 4 Comparative cytoplasmic distribution of TAT-linked recombinant proteins in nondendritic (top) and dendritic cells (bottom, DC). (A) Fluorescence microscopy of living cells incubated for 12 h with red Alexa Fluor 594-conjugated TAT-Ub-PEP proteins. On the right, dark-field fluorescence images were superposed onto phase-contrast bright-field pictures. After incubation with fluorescent proteins, DCs were trypsinized to eliminate the proteins bound to their surface and subjected to cytospin preparation before direct observation without fixation treatment. (B) Low-activity, recombinant TAT–β-galactosidase was added to the culture medium of cells for 16 h and then detected by the X-gal procedure. Cells were photographed under Nomarsky phase contrast. The blue staining is confined to juxtanuclear vesicles in nondendritic cells but appears more diffuse in DCs. DC cultures are contaminated with small round cells corresponding to lymphocytes, barely positive for protein uptake. Molecular Therapy 2005 11, 205-214DOI: (10.1016/j.ymthe.2004.10.010) Copyright © 2004 Terms and Conditions

Fig. 5 In vivo DUB processing of TAT-Ub-Hsp70 monitored by gel fluorimaging. (A) TAT-Ub-Hsp70 recombinant proteins (TUH) were conjugated at the level of the Hsp70 moiety to thiol-reactive Alexa Fluor 488 and added to the culture medium of different cell lines (MCF-7, P19) and of fresh monocyte-derived human dendritic cells (DC). The input fluorescent protein is shown (P). (B) Cleavage of fluorescent TAT-Ub-Hsp70 was compared after incubation with either living U251MG cells or protein extracts from these cells. (C) The cleavage of fluorescent TAT-Ub-Hsp70 was tested after 2-, 6-, or 12-h incubations with DCs. (D) The same experiment as in (C) was done using amine-conjugated fluorescent TAT-Ub-Hsp70. The small fluorescent products yielded after incubation with DCs were resolved on a 16% polyacrylamide gel and analyzed by fluorimaging. Fluorescent ubiquitin (U) and TAT-Ub-PEP (TUP) were run in parallel as molecular weight markers. Control protein extracts from cells incubated with free dye only are shown (lanes C). Molecular Therapy 2005 11, 205-214DOI: (10.1016/j.ymthe.2004.10.010) Copyright © 2004 Terms and Conditions

Fig. 6 Human dendritic cells were incubated or not (0) with TAT-Ub-Bcl-XL proteins (TUB) labeled with succinimidyl ester Alexa Fluor 488, for the indicated periods (5, 20, or 40 min). (A) In addition to intact TUB, fluorimaging of a 15% polyacrylamide SDS–PAGE reveals smaller bands whose molecular weights are compatible with those expected from Bcl-XL alone (25.3 kDa, B) and TAT-Ub (12.5 kDa, TU). As a control, unmodified fluorescent TAT-Ub-Bcl-XL is shown (lane P). (B) The identification of DUB cleavage product in vivo was confirmed by Western probing of the same gel as in (A) using anti-Bcl-XL antibodies. These results are representative of two independent experiments. Molecular Therapy 2005 11, 205-214DOI: (10.1016/j.ymthe.2004.10.010) Copyright © 2004 Terms and Conditions