Junichiro Futami

A New Cytosolic Pathway from a Parkinson Disease-associated Kinase, BRPK/PINK1 ACTIVATION OF AKT VIA MTORC2

Accumulating evidence indicates that dysfunction of mitochondria is a common feature of Parkinson disease. Functional loss of a familial Parkinson disease-linked gene, BRPK/PINK1 (PINK1), results in deterioration of mitochondrial functions and eventual neuronal cell death. A mitochondrial chaperone protein has been shown to be a substrate of PINK1 kinase activity. In this study, we demonstrated that PINK1 has another action point in the cytoplasm. Phosphorylation of Akt at Ser-473 was enhanced by overexpression of PINK1, and the Akt activation was crucial for protection of SH-SY5Y cells from various cytotoxic agents, including oxidative stress. Enhanced Akt phosphorylation was not due to activation of phosphatidylinositol 3-kinase but due to activation of mammalian target of rapamycin complex 2 (mTORC2) by PINK1. Rictor, a specific component of mTORC2, was phosphorylated by overexpression of PINK1. Furthermore, overexpression of PINK1 enhanced cell motility. These results indicate that PINK1 exerts its cytoprotective function not only in mitochondria but also in the cytoplasm through activation of mTORC2.

Truncation of Annexin A1 Is a Regulatory Lever for Linking Epidermal Growth Factor Signaling with Cytosolic Phospholipase A2 in Normal and Malignant Squamous Epithelial Cells

Regulation of cell growth and apoptosis is one of the pleiotropic functions of annexin A1 (ANXA1). Although previous reports on the overexpression of ANXA1 in many human cancers and on growth suppression and/or induction of apoptosis by ANXA1 may indicate the tumor-suppressive nature of ANXA1, molecular mechanisms of the function of ANXA1 remain largely unknown. Here we provide evidence that ANXA1 mechanistically links the epidermal growth factor-triggered growth signal pathway with cytosolic phospholipase A2 (cPLA2), an initiator enzyme of the arachidonic acid cascade, through interaction with S100A11 in normal human keratinocytes (NHK). Ca2+-dependent binding of S100A11 to ANXA1 facilitated the binding of the latter to cPLA2, resulting in inhibition of cPLA2 activity, which is essential for the growth of NHK. On exposure of NHK to epidermal growth factor, ANXA1 was cleaved solely at Trp12, and this cleavage was executed by cathepsin D. In squamous cancer cells, this pathway was shown to be constitutively activated. The newly found mechanistic intersection may be a promising target for establishing new measures against human cancer and other cell growth disorders.

Exploiting protein cationization techniques in future drug development

The development of a method for the efficient intracellular delivery of inherently non-permeable proteins is needed for manipulation of cellular phenotypes or the discovery of protein-based drugs. It has been demonstrated that proteins artificially cationized by chemical conjugation show efficient intracellular delivery via adsorptive-mediated endocytosis and then can exert their biological activity in cells. Studies have also revealed that cationic peptides known as cell-penetrating peptides (CPPs) provide a means to deliver molecules into mammalian cells. Although the internalization mechanisms remain controversial, it is now becoming clear that the main port of entry into cells by CPPs also involves adsorptive-mediated endocytosis rather than the direct penetration of the plasma membrane. As the mammalian cell membrane possesses an abundance of negatively charged glycoproteins and glycosphingolipids, cationization of proteins is a reasonable choice to endow them with the ability for intracellular delivery. Cationization of proteins is usually accompanied by drastic changes in protein properties, structure and biological activities. Recently developed sophisticated protein chemistry can minimize these side effects. Therefore, protein cationization techniques will hopefully prove to be powerful tools for innovative research and drug discovery. In this review, techniques for cationization of proteins and their intracellular delivery, as well as some of their potential therapeutic applications, are discussed.

'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines

A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper‐like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild‐type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L‐RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X‐ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L‐RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L‐ and 2L‐RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L‐RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L‐RNase 1) was also evaluated. N4L‐RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper‐like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.

Dramatic Increase in Expression of a Transgene by Insertion of Promoters Downstream of the Cargo Gene

For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1α, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5′ located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.

Intracellular delivery of glutathione S-transferase-fused proteins into mammalian cells by polyethylenimine-glutathione conjugates.

The glutathione S-transferase (GST)-fused protein expression system has been extensively used to generate a large quantity of proteins and has served for functional analysis in vitro. In this study, we developed a novel approach for the efficient intracellular delivery of GST-fused proteins into living cells to expand their usefulness up to in vivo use. Since protein cationization techniques are powerful strategies for efficient intracellular uptake by adsorptive-mediated endocytosis, GST-fused proteins were cationized by forming a complex with a polycationic polyethylenimine (PEI)-glutathione conjugate. On screening of protein transduction, optimized PEI-glutathione conjugate for protein transduction was characterized by a partly oligomerized mixture of PEI with average molecular masses of 600 (PEI600) modified with multiple glutathiones, which could have sufficient avidity for GST. Furthermore, enhanced endosomal escape of transduced GST-fused proteins was observed when they were delivered with a glutathione-conjugated PEI600 derivative possessing a hydroxybutenyl moiety. These results were confirmed by both intracellular confocal imaging of GST-fused green fluorescent protein and activation of an endogenous growth signal transduction pathway by a GST-fused constitutively active mutant of a kinase protein. These PEI-glutathione conjugates seem to be convenient molecular tools for protein transduction of widely used GST-fused proteins.

Design of cytotoxic ribonucleases by cationization to enhance intracellular protein delivery.

The cytotoxic properties of naturally occurring or engineered RNases correlate well with their efficiency of cellular internalization and digestion level of cellular RNA. Cationized RNases are considered to adsorb to the anionic cellular surface by Coulombic interactions, and then become efficiently internalized into cells by an endocytosis-like pathway. The design of cytotoxic RNases by chemical modification of surface carboxylic residues is one of the powerful strategies for enhancing cellular internalization and is accompanied with a decreased sensitivity for the cytoplasmic RNase inhibitor. Although chemically modified cationized RNases showed decreased ribonucleolytic activity, improved endocytosis and decreased affinity to the endogenous RNase inhibitor conclusively contribute to their ability to digest cellular RNA. Furthermore, the cytotoxicity of cationized RNases can be drastically enhanced by co-endocytosis with an endosome-destabilizing peptide. Since efficient cellular internalization of proteins into living cells is an important technology for biotechnology, studies concerning the design of cytotoxic RNases provided general perceptions for protein-based drug design.

A novel gene expression system strongly enhances the anticancer effects of a REIC/Dkk-3-encoding adenoviral vector

Gene expression systems with various promoters, including the cytomegalovirus (CMV) promoter, have been developed to increase the gene expression in a variety of normal and cancer cells. In particular, in the clinical trials of cancer gene therapy, a more efficient and robust gene expression system is required to achieve sufficient therapeutic outcomes. By inserting the triple translational enhancer sequences of human telomerase reverse transcriptase (hTERT), Simian virus 40 (SV40) and CMV downstream of the sequence of the BGH polyA, we were able to develop a novel gene expression system that significantly enhances the expression of the genes of interest. We termed this novel gene expression cassette the super gene expression (SGE) system, and herein verify the utility of the SGE cassette for a replication-deficient adenoviral vector. We newly developed an adenoviral vector expressing the tumor suppressor, reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3), based on the CMV promoter-driven SGE system (Ad-SGE-REIC) and compared the therapeutic utility of Ad-SGE-REIC with that of the conventional adenoviral vectors (Ad-CMV-REIC or Ad-CAG-REIC). The results demonstrated that the CMV promoter-SGE system allows for more potent gene expression, and that the Ad-SGE-REIC is superior to conventional adenoviral systems in terms of the REIC protein expression and therapeutic effects. Since the SGE cassette can be applied for the expression of various therapeutic genes using various vector systems, we believe that this novel system will become an innovative tool in the field of gene expression and gene therapy.

Molecular cloning and expression of human ribonuclease 4 cDNA

A cDNA coding for human ribonuclease 4 was isolated from a pancreas cDNA library and sequenced. This cDNA (996 bp) includes an entire open reading frame encoding mature protein (119 aa) following signal peptide (28 aa). Expression of mature protein in Escherichia coli showed an apparent molecular mass of about 16 kDa, which was slightly lower than the mature form of human RNase 1, in SDS-PAGE.

Leucyl/Phenylalanyl-tRNA-Protein Transferase-Mediated Chemoenzymatic Coupling of N-Terminal Arg/Lys Units in Post-translationally Processed Proteins with Non-natural Amino Acids

Position-specific incorporation of non-natural amino acids through ribosomal systems can introduce functional groups into proteins at desired positions. Through the use of multiple four-base codons, we have successfully incorporated two or even three non-natural amino acids into single proteins in vitro. Because at least five mutually orthogonal four-base codons have been identified, up to five non-natural amino acids can, in theory, be incorporated into single proteins, but the limited decoding efficiency of the four-base codons means that triple mutation is the current limit. Further incorporation of non-natural amino acids into specific positions of proteins needs another technique. We and other groups have introduced a strategy for the biosynthetic incorporation of a probe only at the N terminus of a nascent protein. At this stage, an E. coli aminoacyl-tRNA synthetase mixture was used to charge initiator methionine tRNA with methionine, and the a-amino group of the MettRNA was then treated with an amine-reactive chemical probe. In this way the probe could be incorporated at the Nterminal ends of several proteins, but the approach is currently limited by low incorporation efficiency and by the production of only limited quantities of the labeled protein in a batch process. Enzymatic modification of proteins without the participation of ribosomes is an alternative approach to the position-specific introduction of functional groups. Leucyl/phenylalanyl ACHTUNGTRENNUNG(L/F)tRNA-protein transferase from E. coli is known to catalyze the transfer of hydrophobic amino acids—such as phenylalanine, leucine, or methionine—from tRNA to the N termini of proteins possessing lysine or arginine as their N-terminal residues. N-terminal arginine and lysine are secondary destabilizing residues in E. coli because the destabilization depends on their conjugation to the leucine or phenylalanine primary destabilizing residues by the transferase. Here we report that this enzymatic coupling can be expanded to link non-natural amino acids to the N termini of target proteins through the use of tRNAs aminoacylated with various types of non-natural amino acid. Because this new technique can be used independently of non-natural amino acid mutagenesis, it provides another method through which to add a single extra non-natural amino acid, in addition to the incorporation of multiple non-natural amino acids through orthogonal four-base codons. Proteins expressed in bacteria normally contain methionine units at their N termini, except in cases in which the next amino acid is a small one, so we developed a novel and convenient procedure to attach single Lys units to the N termini of proteins, choosing SoCBM13—known as a xylan-binding domain (XBD)—as our target protein. We cloned the SoCBM13 coding sequence into a pET27 plasmid vector to obtain an expressing plasmid pET27-Lys-SoCBM13. The vector contains a pelB signal peptide coding region directly before the cloning site (see Supporting Information). By introducing this plasmid into E. coli we expressed a fusion protein of pelB signal peptide and Lys-SoCBM13. After translation, the pelB signal peptide is spontaneously cleaved off in E. coli, leaving a single Lys unit and providing the desired Lys-SoCBM13. As a negative control, we also prepared His-SoCBM13, in which a single histidine unit is attached to the N terminus. Successful syntheses of the extended SoCBM13 were confirmed by TOF-MS, as ACHTUNGTRENNUNGdescribed in the Supporting Information. Initially we chose 3-nitrotyrosine as the non-natural amino acid, as it can easily be detected by use of an anti-nitrotyrosine antibody in Western blotting. E. coli tRNA was aminoacylated with nitrotyrosine with use of a resin-supported ribozyme for aminoacylation and the resulting ntrTyr-tRNA was added to a reaction mixture containing the extended SoCBM13 and the transferase. The products were analyzed by Western blotting and visualized with the anti-nitrotyrosine antibody (Figure 1). A band containing the ntrTyr unit was detected only in the presence of Lys-SoCBM13 and ntrTyr-tRNA (lane 1), while no ntrTyr-linked protein was detected in the mixture of HisSoCBM13 and ntrTyr-tRNA (lane 2), or in the mixture of LysSoCBM13 and free tRNA (lane 3). These results clearly indicate that the non-natural amino acid had successfully been coupled to Lys-SoCBM13. The presence and the position of the ntrTyr unit were examined by a sequence analysis of the product based on Edman [a] Prof. Dr. M. Taki, S. Matoba, Y. Kobayashi, Prof. Dr. J. Futami, Prof. Dr. M. Sisido Department of Bioscience and Biotechnology Faculty of Engineering, Okayama University 3-1-1 Tsushimanaka, Okayama 700-8530 (Japan) Fax: (+81)86-251-8219 E-mail : taki@biotech.okayama-u.ac.jp sisido@cc.okayama-u.ac.jp [b] Prof. Dr. M. Taki, Dr. A. Kuno, Prof. Dr. K. Taira National Institute of Advanced Industrial Science and Technology (AIST) 1-1-1 Higashi, Tsukuba Science City, 305-8562 (Japan) [c] Prof. Dr. H. Murakami, Prof. Dr. H. Suga, Prof. Dr. K. Taira Department of Chemistry and Biotechnology Graduate School of Engineering, Tokyo University Hongo, Tokyo 113-8656 (Japan) [d] Prof. Dr. T. Hasegawa Department of Material and Biological Chemistry Faculty of Science, Yamagata University Yamagata 990-8560 (Japan) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.