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A cutinase-like enzyme from a thermophilic isolate, Saccharomonospora viridis AHK190, Cut190, has the ability to depolymerize polyethylene terephthalate (PET). The catalytic activity and thermal stability of Cut190 are increased by Ca2+ binding. The structural analysis of Cut190 mutants in complex with metal ions and substrates elucidated the reaction mechanism regulated by Ca2+. The metal ion-binding properties, analyzed using isothermal titration calorimetry were correlated with the effects on Cut190 activity and stability, which could be improved using protein engineering. The Cut190 mutant will be used for PET chemical recycling. [ABSTRACT FROM AUTHOR]

Protein design holds promise for applications such as control of cells, therapeutics, new enzymes and protein-based materials. Recently, rational design of protein molecules has made a great progress, guided by the consistency principle proposed by Nobuhiro Gō in 1983: local and non-local interactions consistently favor the same folded conformation. We discovered a set of rules for designing ideal protein structures stabilized by consistent local and non-local interactions. The rules enabled the de novo design of amino acid sequences having the funnel-shaped energy landscapes toward the desired target structures. Various ideal protein structures have been created using the rules. [ABSTRACT FROM AUTHOR]

Since the discovery of channelrhodopsins in 2002-2003, the light is now engineered to manipulate physiological states of a cell, a tissue or a body with the progress of three kinds of researches: (1) molecular biology of photoreceptive proteins, (2) cause-effect relationships in the biological system and (3) fabrication of opto-electronics or nanodevices. The near-infrared light, which conveys energy deep into the tissues, is expected to be used for optogenetics with the creation of proteins that change their conformations with such energy or the improvement of nanodevices that transfer the energy to photoreceptive proteins. [ABSTRACT FROM AUTHOR]

Selective protein labeling enables us to visualize and engineer proteins in living cells. We developed chemical labeling methods for proteins in test tubes as well as in living cells based on ligand-directed chemistry. Using these methods, small functional molecules can be modified in the vicinity of the ligand binding pocket of target proteins with restoring the protein function. Here we overview our strategies and introduced their applications. [ABSTRACT FROM AUTHOR]

Growth factors had been believed to act on cells only in the soluble stat. However, during two decades researchers demonstrated that growth factors bound on solid substrata also act on cells. In addition, the details of mechanism have been studied. In this article, the action mechanism of bound growth factors will be discussed. Subsequent creation and application of newly developed growth factors, which have binding affinities, will be renewed. Collagen binding domain-containing epidermal growth factor (EGF) or hepatocyte growth factor, fibrin binding domain-containing EGF, and substrate-binding growth factors were synthesized and some were applied for animal model experiments. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-α (TNF), a proinflammatory cytokine, is critical to the pathogenesis of various inflammatory diseases. There are two subtypes of receptors for TNF, namely type I TNF receptor (TNFR1) and type II TNF receptor (TNFR2). Previous studies using animal models of diseases have demonstrated the predominant role of TNFR1 in the pathogenesis of inflammation. It has recently been proposed that TNFR2 is associated with anti-inflammatory function. This intriguing function of TNFR2 has implications from an immunological and pharmacological perspective. However, the mechanism of the TNFR2-mediated anti-inflammatory effect is not fully understood. In this context, we attempted to elucidate the TNFR2-mediated anti-inflammatory effect and other unknown biological functions of TNFR2 by utilizing our protein engineering technology to generate functional mutant cytokines. Our findings reveal the following. (1) TNFR2 is expressed on regulatory T cells (Tregs) but not conventional T cells (Tconvs) and TNFR2-mediated signals promote proliferation and activation of Tregs. (2) The crystal structure of TNF/TNFR2 complex was solved, which suggests a possible signal initiation mechanism via TNF/TNFR2 cluster formation on the cellular membrane. (3) A novel TNFR2-mediated signal molecule, aminopeptidase P3 (APP3/XPNPEP3), was identified that interacts with TNFR2 as an intracellular adaptor protein. APP3 is required for c-Jun N-terminal kinase (JNK) phosphorylation, the downstream molecule of TNFR2 signal transduction. These results are key to understanding the mechanism of immune regulation and will assist in the identification of immunomodulatory drugs targeting the TNFR2 signaling cascade as well as the function of Tregs.

Proteins are excellent materials for constructing nano- to micro-meter sized compartments. For example, in nature, hollow spherical shells made of proteins, called protein cages, are widespread. Prominent examples include viruses, ferritins, carboxysomes, and others. Protein cages designed and engineered in the laboratory have also gained recent attention because of their potential use in synthetic biology, materials science, and medicine. Here, we show that engineered variants of lumazine synthase (LS) from Aquifex aeolicus self-assemble into porous shell-like structures, with striking size-expansion from the original dodecahedron composed of 12 pentamer subunits. Cryo-electron microscopy (EM) analysis has revealed that pentamers are the basic assembly units, although small conformational changes in each subunit lead to final expanded architectures composed of 36 and 72 pentamers. The underlying conformational changes likely arise from electrostatic repulsion between anionic residues originally introduced at the lumenal surface of the LS cage to encapsulate positively charged guest molecules. The plastic nature of the LS cage structure was also explored using a positively supercharged variant of the green fluorescent protein GFP(+36) as an assembly mediator. By controlling the favorable electrostatic interactions between the negatively charged LS cage and the positively charged mediator, multishell structures were created, as previously observed in some virus-like particles. These results highlight the potential of engineered LS cages for various future applications, including drug delivery and bioimaging.

Immune-checkpoint inhibitors have shown their efficacy in the treatment of patients with many kinds of progressive/relapsed cancers. However, the efficacy remains as 10-40%of the patients in most type of cancers, suggesting that the development of new therapy for patients resistant to the therapy is an urgent unmet need. Adoptive therapy with tumor-specific T cells is a promising therapy that can be effective in patients who are not benefited from the immune-checkpoint inhibitors. The T cell therapy with genetic engineering in T cell receptor(TCR)is expected to be a universal therapy because this therapy can be applicable for patients with many kinds of cancers. This review outlines the recent clinical development of cancer immunotherapy utilizing TCR-gene engineered T cells and discusses the issues that should be improved in the near future for the safer and more effective TCR-T cell therapy of the broad range of cancer patients.

Although antibodies have been used as molecularly targeted agents for difficult-to-treat diseases such as cancers, the high production costs associated with mammalian expression systems continue to be a drawback. In addition, the clinical efficacy of conventional IgG antibodies is limited. Several types of recombinant antibody (e.g. fused with anticancer drugs, multivalent, or multispecific) have been designed in efforts to develop next-generation antibodies with higher functionality. We used protein engineering to construct several anticancer recombinant antibodies by developing bispecific antibodies that induced specific antitumor effects against cancer cells through the recruitment of lymphocytes. We found that a humanized small bispecific antibody (Ex3) that targets epidermal growth factor receptor on tumor cells and CD3 on T lymphocytes had marked anticancer activity. Furthermore, the function of Ex3 was enhanced by fusion with the human Fc region, domain rearrangement, multimerization, and affinity maturation; a combination of these modifications showed at least additive cytotoxic effects. Interestingly, merely rearranging the domain order of Ex3 induced substantial cytotoxic enhancements, even though the structural format remained the same. Here, we describe our efforts to develop highly functional bispecific antibodies as next-generation therapeutic antibodies using protein engineering.

An understanding of the physiological significance of peptides and proteins is indispensable in the fields of life sciences and drug development. Recently, methods for controlling peptide and protein activities using stimuli such as UV irradiation have been attracting much attention because of their potential for clarifying the physiological roles of the peptides/proteins. In this context, we have developed a stimulus-responsive amino acid that induces peptide-bond cleavage after exposure to a stimulus. Although it has previously been reported that stimulus-responsive units can respond to a specific stimulus, our stimulus-responsive amino acid is potentially applicable to any stimulus simply by changing the protective group. In this review, the design and synthesis of stimulus-responsive amino acids are described. Their applications in chemical biology, including their use for spatiotemporal control of the activity of peptides in living cells, are also reported.

The trace characterization of physiologically active substances with low molecular weight (e.g., steroids, catecholamines, prostaglandins, and oligopeptides), which are classified as "haptens", is an important subject in clinical analysis, and competitive immunoassays have conventionally been used for this purpose. However, the subfemtomole-range determination of haptens is very difficult, as the sensitivity of competitive immunoassays is essentially limited by the affinity of the anti-hapten antibodies that barely reaches the range of 10(11) (l/mol) as the affinity constant (K(a)). Although a noncompetitive "immunometric assay" format, the two-site immunometric assay (sandwich immunoassay), enables even subattomole-range measurements of macromolecules such as proteins, this principle can not be directly applied to haptens, as their low molecular mass prohibits simultaneous binding by two antibody molecules. To overcome such limitations, we are required either to create artificial antibodies showing ultrahigh affinity to haptens by protein engineering of antibody molecules ("antibody engineering") or establishment of novel immunometric assay formats applicable to haptens. This review surveys the background and recent approach for subfemtomole-range determination of haptens using novel immunometric assay methods. Our studies for the development of hapten immunometric assays are also described.

Proteins having multiple epitopes can be usually measured with sandwich ELISA employing two kinds of antibodies, which permits high sensitivity as well as a wide working range of more than three orders of magnitude. On the other hand, so-called monovalent antigens with MW less than 1000 are not susceptible to sandwich assays due to their small size and have almost always been measured in competitive assays. However, while a competitive assay needs only one antibody due to the principle of ratiometric measurement, optimization of the reaction conditions is necessary to attain suitable sensitivity and working range, which are often inferior to those of sandwich assays. As an alternative immunoassay for small antigens, here we propose a noncompetitive "open sandwich" immunoassay, which is based on the principle of stabilization of the antibody variable region Fv upon binding with antigen. Using ELISA to detect labeled V(H) fragments bound to immobilized V(L) in the presence of sample in microplate wells, various small molecules with MW around 200-300 can be measured with a superior detection limit and working range compared with those achieved with the corresponding competitive assays. The results indicate a common antigen recognition mode of anti-hapten antibody and wide applicability of the assay to the sensitive and handy analysis of low molecular-weight substances in areas such as environmental analysis and clinical diagnostics.

The sequence of the human genome has been decoded and a post-genome era is now beginning. This is the start of the protein/peptide era. Working in the fundamental research division of a pharmaceutical company and being engaged in studies on the research and development of new drugs based upon genomic information, it was clear to me that genomic information was useful, significant and indispensable in driving forward to new drug discovery. However, it is also true that we need more than that. I have faced multiple barriers in pursuing genomic information alone. There are numerous dead-end stories in genome-based new drug discovery and in many cases, those hurdles are very hard to get over. What approach can be a breakthrough of this bottleneck? I believe that one answer is research into proteins. The proteome describes all the proteins within an organism. The proteome analysis research field promises a bright future for the discovery of new drugs, diagnosis and therapeutics. I believe the mass spectrometer(MS) will be a key instrument in this research. The contribution of mass spectrometry was recently recognized in the Nobel Prize for Chemistry 2002, for 'the discovery of Soft Laser Desorption/Ionization' awarded to Mr. Koichi Tanaka. There is no doubt that the MS will play an essential role in accelerating proteome analysis. However, the present proteome analysis technology has not yet reached a sufficient scientific level. Further improvements of both hardware and software are necessary. For the hardware, improvements in sensitivity, accuracy and high-throughput, in addition to equipment for ultra-micro analysis applied to the analysis of the proteome should be addressed. Regarding software, we need to develop new chemistries for proteome analyses and propose original, user-friendly proteome analysis methods. In this review, starting with a briefing on genome-based drug discovery, I will discuss a new concept for proteome analysis based on mass spectrometry and new methodologies. This 'next generation' platform may provide a solution for the development of an integrated system that will contribute to our understanding of clinical information/bioinformatics, therapeutics, diagnosis and even drug discovery.

I have pursued research on lysozymes for 42 years. During that time, I made Several new findings, some of them by chance. My enjoyment of the following areas is reviewed: the story of tryptophan; protease digestion mechanisms; peptide mapping with RP-HPLC; gene engineering; renaturation of protein; catalytic residues; fluctuation and function; stabilization; folding; antigenecity; tolerance; and various lysozymes.

This paper reviews the results of basic research conducted by the author's group to determine appropriate methods to develop protein-based drugs. These include production strategies, elucidation of physiologic function, improving existing pharmaceuticals, de novo design, and protein reconstruction. The antigenicity of modified proteins and methods to induce antigenic protein tolerance are also described.

Respiratory syncytial virus remains a significant cause of severe lower respiratory tract disease in children. The risk of serious RSV illness is highest among children with prematurity, chronic lung disease and congenital heart disease. No effective vaccine and anti-viral agents have been obtained even now. Therefore, conservative therapy including respiratory aid has been a principal therapy for serious RSV disease. Recently, monthly intramuscular administration of humanized anti-RSV monoclonal antibody(palivizumab) was introduced in clinical fields in USA and Europe. Palivizumab prophylaxis has appeared to be safe and effective for prevention of serious RSV illness in premature children and those with bronchopulmonary dysplasia.

In rheumatoid arthritis and inflammatory bowel disease, such as Crohn's disease, certain immunological abnormalities are considered as its cause, but the fundamental mechanism remains unclear. However, recent researches revealed that inflammatory cytokines, such as TNF-alpha, IL-1 and IL-6, are greatly involved in these immunological abnormalities. This led to the development of anti-cytokine therapy using monoclonal antibodies to these cytokines. Among them, Infliximab, a chimeric monoclonal antibodies to TNF-alpha, is most clinically applied and marked therapeutic effect is seen in various diseases.

Antibodies can have a medicinal role because of their high specificity in molecular recognition. Although monoclonal antibodies specific for an antigen can be obtained relatively easily, it is difficult to acquire ones with high affinity. Recently, antibody engineering using an in vitro evolutionary process has been put to this task. However, little progress seems to have been made. Our previous study revealed that antibodies have low or high evolvability. An antibody with a poor ability to evolve would also not attain high affinity in in vitro maturation. In this review, we provide information for the improved design of the antigen-combining site and that this will contribute to the engineering of antibodies using an in vitro evolutionary process.

Since the discovery of monoclonal antibody(MoAb) in 1975, MoAbs have held great promise for the treatment of human disease such as cancer, viral infection, and autoimmune disorders. The most important limitations in murine MoAb have been the immune response against murine immunoglobulins and insufficient activation of human effector function. The emergence of antibodies as an attractive therapy is the result of the evolution of MoAb technology over the past 25 years. These problems have been overcome using genetic engineering techniques to produce chimeric mouse-human, CDR-grafted, and fully human antibodies. New strategies to develop chimeric, humanized, and completely human antibodies are discussed in this review.

1. In order to understand the differences in pH optima and reaction rates of RNase A towards low molecular weight substrates and polymer substrates, the subsite structure of bovine pancreatic RNase A was studied. The kinetic studies of various sizes of oligouridylic acids showed that the size of the subsite is three nucleotides long. The kinetic studies on the inhibition of pUp, X-ray crystallographies of RNase A-ApC and pTp complexes, 31P-NMR studies on the binding of RNase A-pAp, and pTp showed the presence of P0, P2 and B3 sites. The location of the P0 site was assigned to be Lys66 by X-ray crystallography of the RNase A-pTp complex. The location of the P2 and/or P3/B3 site was determined by studying the enzymatic activities of several S-peptide analogs in which N-Leu was substituted for Lys7 and/or Lys1 coupled with S-protein toward various chain lengths of oligouridylic acids. The experiment suggested that P2 is Lys7 and P3/B3 is Lys1. 2. Several new pyrimidine base specific RNases were isolated and their primary structures were determined. They were two non-secretory RNases, a bovine liver alkaline RNase, a bovine brain RNase, and a bullfrog liver RNase. The bovine brain RNase has extra 16 amino acids at the C-terminus with O-glycosylated Ser. The bullfrog liver RNase was an extremely heat-stable RNase so far known. 3. Two new RNases belonging to RNase T1 family were isolated and their primary structures were elucidated. They were RNases isolated from Aspergillus saitoi and a mushroom (hiratake). The former RNase has a similar structure to RNase T1, but it was a base non-specific and guanylic acid preferential enzyme. From the results of X-crystallographic studies of this RNase, we suggested that the mechanism of RNase T1 RNase is essentialy a general acid-base catalysis between His40 and Glu58. 4. We isolated several fungal, plant and animal base non-specific acid RNases with a molecular mass about 24 kDa or more, and elucidated their primary structures. These RNases contain two sequences containing common 7-8 amino acid residues in common which include most of the amino acid residues important for the catalysis. Therefore, we proposed to designate these RNases as RNase T2 family RNase. On the basis of chemical modifications, kinetic studies and protein engineering studies of RNase Rh from Rhizopus niveus and RNase M from A. saitoi, we assigned that the catalytic site of RNase Rh consists of His46, His104, His109, Glu105, and Lys108. In the mechanism we proposed for RNase Rh, His46 and His109 work as a general acid and base catalysts. His104 was a phosphate binding site, and Glu105 and Lys108 might work to polarize a P=O bond of the substrate or stabilize the pentacovalent intermediate. However, in the reverse reaction of the transfer reaction step and the hydrolysis step of RNase Rh, His109 and His46 work as an acid and base catalyst, respectively. The X-ray crystallographic studies of RNase Rh, an RNase Rh-2'-AMP or d(ApC)complex, and the protein engineering studies of several mutant enzymes assigned the components of the major base recognition site (B1 site) and the minor base recognition site (B2 sites) of RNase Rh. The enzymatic studies of several mutant enzymes indicated that (i) Asp51 is very crucial for adenine base recognition, and the replacement of Asp51 by other amino acid, such as Thr, Ser, Glu, Asn makes RNase Rh more guanylic acid preferential, (ii) the replacement of Trp49 by Phe, and Tyr57 by Trp make the enzyme more pyrimidine and purine bases preferential, respectively. These trials are the first example of marked artificial change in the base specificity of RNases.

Proteins are usually in an equilibrium between the folded and the unfolded state. Therefore, for the stabilization of proteins against reversible denaturation, the free energy change for the unfolding should be increased by stabilizing the folded state by lowering the energy level of the folded state or by destabilizing the unfolded state by raising the energy level of the unfolded state. On the other hand, various processes can be coupled with the unfolded state of proteins. For example, protease digestion of proteins at physiological temperature may be one of such processes. The process would lead to an irreversible denaturation. For the stabilization of proteins against the irreversible denaturation coupled with the unfolded state, a kinetic stabilization is important, that is, the activation free energy for the unfolding should be increased. Methods for the kinetic stabilization were discussed. Finally, the irreversible chemical deterioration of proteins was considered.