Uniformly cationized protein efficiently reaches the cytosol of mammalian cells.

Protein cationization techniques are powerful protein transduction methods for mammalian cells. As we demonstrated previously, cationized proteins with limited conjugation to polyethylenimine have excellent ability to enter into cells by adsorption-mediated endocytosis [Futami, J., et al. (2005) J. Biosci. Bioeng. 99, 95-103]. In this study, we show that proteins with extensive and uniform cationization covering the protein surface reach the cytoplasm and nucleus more effectively than proteins with limited cationic polymers or proteins that are fused to cationic peptides. Although extensive modification of carboxylates results in loss of protein function, chicken avidin retains biotin-binding ability even after extensive amidation of carboxylates. Using this cationized avidin carrier system, the protein transduction ability of variously cationized avidins was investigated using biotinylated protein as a probe. The results revealed that cationized avidins bind rapidly to the cell surface followed by endocytotic uptake. Small amounts of uniformly cationized avidin showed direct penetration into the cytoplasm within a 15 min incubation. This penetration route seemed to be energy dependent and functioned under cellular physiological conditions. A biotinylated exogenous transcription factor protein that penetrated cells was demonstrated to induce target gene expression in living cells.

Cryopreservation of rat islets of Langerhans by vitrification.

Cryopreservation could be a possible means of addressing the shortage of islets of Langerhans. We investigated the effects of EDT324 solution on the vitrification of isolated rat islets of Langerhans. Rat pancreatic islets were cryopreserved in 10% DMSO by a slow-rate freezing method or were cryopreserved in EDT324 solution by vitrification. The cryopreserved islets were compared in terms of viability, stimulation index and metabolic function after transplantation. After cryopreservation, the viability and stimulation of islets stored in EDT324 were 92.4% and 6.4, respectively, and were higher than islets stored by slow freezing (72.5% and 1.5, respectively). Streptozotocin-induced diabetic rats were transplanted with islets cryopreserved in EDT324, which corrected diabetes and achieved euglycemia within 2 days after transplantation. These results indicate that EDT324 allows successful cryopreservation of rat islets for long-term storage as an alternative solution to traditionally used solutions, such as 10% DMSO. Transplantation of cryopreserved islets into diabetic rats can achieve euglycemia.

Quantitative screening of EGF receptor-binding peptides by using a peptide library with multiple fluorescent amino acids as fluorescent tags.

EGF receptor-binding peptides could be found by a peptide screening method using fifteen fluorescent amino acids as fluorescent tags. Of 225 peptides, we found an 8-mer peptide containing a dipeptide unit, Y-F, which was the strongest binding peptide to the EGF receptor.

Evaluation of irreversible protein thermal inactivation caused by breakage of disulphide bonds using methanethiosulphonate.

Many extracellular globular proteins have evolved to possess disulphide bonds in their native conformations, which aids in thermodynamic stabilisation. However, disulphide bond breakage by heating leads to irreversible protein denaturation through disulphide-thiol exchange reactions. In this study, we demonstrate that methanethiosulphonate (MTS) specifically suppresses the heat-induced disulphide-thiol exchange reaction, thus improving the heat-resistance of proteins. In the presence of MTS, small globular proteins that contain disulphides can spontaneously refold from heat-denatured states, maintaining wild-type disulphide pairing. Because the disulphide-thiol exchange reaction is triggered by the generation of catalytic amounts of perthiol or thiol, rapid and specific perthiol/thiol protection by MTS reagents prevents irreversible denaturation. Combining MTS reagents with another additive that suppresses chemical modifications, glycinamide, further enhanced protein stabilisation. In the presence of these additives, reliable remnant activities were observed even after autoclaving. However, immunoglobulin G and biotin-binding protein, which are both composed of tetrameric quaternary structures, failed to refold from heat-denatured states, presumably due to chaperon requirements. Elucidation of the chemical modifications involved in irreversible thermoinactivation is useful for the development of preservation buffers with optimum constitutions for specific proteins. In addition, the impact of disulphide bond breakage on the thermoinactivation of proteins can be evaluated using MTS reagents.

Mitochondrial determinants of mammalian longevity.

Current ageing theories are far from satisfactory because of the many determinants involved in ageing. The well-known rate-of-living theory assumes that the product (lifetime energy expenditure, LEE) of maximum lifespan (MLS) and mass-specific basal metabolic rate (msBMR) is approximately constant. Although this theory provides a significant inverse correlation between msBMR and MLS as a whole for mammals, it remains problematic for two reasons. First, several interspecies studies within respective orders (typically within rodents) have shown no inverse relationships between msBMR and MLS. Second, LEE values widely vary in mammals and birds. Here, to solve these two problems, we introduced a new quantity designated as mitochondrial (mt) lifetime energy output, mtLEO = MLS × mtMR, in place of LEE, by using the mt metabolic rate (mtMR) per mitochondrion. Thereby, we found that mtLEO values were distributed more narrowly than LEE ones, and strongly correlated with the four amino-acid variables (AAVs) of Ser, Thr and Cys contents and hydrophobicity of mtDNA-encoded membrane proteins (these variables were related to the stability of these proteins). Consequently, only these two mt items, mtMR and the AAVs, solved the above-mentioned problems in the rate-of-living theory, and thus extensively improved the correlation with MLS compared with that given by LEE.

Enhanced in-cell folding of reversibly cationized transcription factor using amphipathic peptide.

The intracellular delivery of functionally active transcription factor proteins is emerging as a promising technique for artificial regulation of cellular functions. However, in addition to the cell membrane, which acts as a barrier to macromolecules, the aggregation-favored properties of structurally flexible transcription factor proteins limit the application of this method. In-cell folding technique can be used to overcome these issues. This technique solubilizes denatured protein by reversible alkyl-disulfide cationization (S-cationization), and simultaneously endows efficient intracellular delivery and folding to the biologically active conformation in the reducing environment of the cytosol. Because cationized protein is internalized into cells by adsorption-mediated endocytosis, endosomal escape is crucial for this technique. In this study, we utilized a sensitive luciferase reporter gene assay to quantitatively evaluate in-cell folding of the artificial transcription factor GAL4-VP16. Although the cationic moiety of S-cationized protein was slightly affected, co-transduction of amphipathic peptide Endo-PORTER dramatically improved in-cell folding efficiency. Live cell imaging of fluorescent-labeled GAL4-VP16 revealed that some of the proteins diffused into the cytosol and nucleus through co-transduction with Endo-PORTER. Real-time monitoring of light output of luciferase revealed the kinetics of in-cell folding, supporting that endosomal-release assisted by Endo-PORTER was stimulated by endosome acidification. Because this method can transduce proteins uniformly and repeatedly into living cells, S-cationized transcription factor proteins are widely applicable for the artificial regulation of cellular functions.

A novel method for screening peptides that bind to proteins by using multiple fluorescent amino acids as fluorescent tags.

We describe a new screening method for simultaneously detecting peptides that bind to a target protein by fluorescence obtained from fluorescent amino acid-modified peptides.