Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer.

TFF2 is a small, secreted protein with anti-inflammatory properties. We previously have shown that TFF2 gene delivery via adenovirus (Ad-Tff2) suppresses colon tumor growth in colitis associated cancer. Therefore, systemic administration of TFF2 peptide could potentially provide a similar benefit. Because TFF2 shows a poor pharmacokinetic, we sought to modify the TFF2 peptide in a manner that would lower its clearance rate but retain bioactivity. Given the absence of a sequence-based prediction of TFF2 functionality, we chose to genetically fuse the C-terminus of TFF2 with the carboxyl-terminal peptide of human chorionic gonadotropin ß subunit, and inserted into adenoviral vector that expresses Flag. The resulting Ad-Tff2-CTP-Flag construct translates into a TFF2 fused with two CTP and three Flag motifs. Administered Ad-Tff2-CTP-Flag decreased tumorigenesis and suppressed the expansion of myeloid cells in vivo. The fusion peptide TFF2-CTP-Flag delivered by adenovirus Ad-Tff2-CTP-Flag as well purified recombinant fusion TFF2-CTP-Flag was retained in the blood longer compared with wild-type TFF2 delivered by Ad-Tff2 or recombinant TFF2. Consistently, purified recombinant fusion TFF2-CTP-Flag suppressed expansion of myeloid cells by down-regulating cyclin D1 mRNA in vitro. Here, we demonstrate for the very first time the retained bioactivity and possible pharmacokinetic advantages of TFF2 with a modified C-terminus.

Delineating the extracellular water-accessible surface of the proton-coupled folate transporter.

The proton-coupled folate transporter (PCFT) was recently identified as the major uptake route for dietary folates in humans. The three-dimensional structure of PCFT and its detailed interplay with function remain to be determined. We screened the water-accessible extracellular surface of HsPCFT using the substituted-cysteine accessibility method, to investigate the boundaries between the water-accessible surface and inaccessible buried protein segments. Single-cysteines, engineered individually at 40 positions in a functional cysteine-less HsPCFT background construct, were probed for plasma-membrane expression in Xenopus oocytes with a bilayer-impermeant primary-amine-reactive biotinylating agent (sulfosuccinimidyl 6-(biotinamido) hexanoate), and additionally for water-accessibility of the respective engineered cysteine with the sulfhydryl-selective biotinylating agent 2-((biotinoyl)amino)ethyl methanethiosulfonate. The ratio between Cys-selective over amine-selective labeling was further used to evaluate three-dimensional models of HsPCFT generated by homology / threading modeling. The closest homologues of HsPCFT with a known experimentally-determined three-dimensional structure are all members of one of the largest membrane protein super-families, the major facilitator superfamily (MFS). The low sequence identity--14% or less--between HsPCFT and these templates necessitates experiment-based evaluation and model refinement of homology/threading models. With the present set of single-cysteine accessibilities, the models based on GlpT and PepTSt are most promising for further refinement.