Thermodynamic analysis of a multifunctional RNA-binding protein, PhoRpp38, in the hyperthermophilic archaeon Pyrococcus horikoshii OT3

The protein component PhoRpp38 of Pyrococcus horikoshii ribonuclease P (RNase P) is known to be a multifunctional RNA-binding protein. Previous biochemical data indicate that it binds to two stem-loops in RNase P RNA (PhopRNA). Thermodynamic analysis revealed that PhoRpp38 and PhopRNA interact with each other with an association constant (Ka) of 1.56×10(7) M(-1). It was further found that PhoRpp38 simultaneously binds two stem-loop structures in PhopRNA with approximately equal affinity. Crystals of PhoRpp38 in complex with the stem-loop were grown and diffracted to a resolution of 7.0 ? on a synchrotron X-ray source.     

Efficient expression of recombinant soluble human FcγRI in mammalian cells and its characterization

The extracellular domain of human FcγRI which interacts with a human IgG was expressed as recombinant soluble human FcγRI (rshFcγRI) by Chinese hamster ovary (CHO) cell. Stable CHO cell clones with efficient expression of rshFcγRI were established based on a dihydrofolate reductase (DHFR)/methotrexate (MTX) gene-amplification system. The CHO clones efficiently produced rshFcγRI under high-density continuous culture in a bioreactor. After 53 days of culture, the number of cells had reached approximately 4 × 10? cells/mL in the bioreactor and the average production of rshFcγRI had reached 7.4 mg L-medium?1 day?1. Secreted rshFcγRI was purified to a homogeneous state using cation exchange and affinity chromatographies. The binding affinities of rshFcγRI to human IgG subclasses were determined using surface plasmon resonance analysis. The binding affinities of rshFcγRI to human IgG1/κ and IgG3/κ were high (1.59 × 10?1? and 2.81 × 10?1? M, respectively), whereas that of rshFcγRI to human IgG4/κ was lower binding affinity (1.41 × 10?? M). Binding to IgG2/κ was not detectable. Examination of circular dichroism spectra indicated that rshFcγRI was rich in β-structures and loop or turn structures, but there were few α-helices. These results may be valuable for further studies of the structure and function of human FcγRI.     

Structural and thermodynamic characterization of the self-adhesive properties of human P-cadherin

Human P-cadherin is a promising therapeutic target against cancer. However, its characterization at the molecular level is still lacking. We report that human P-cadherin associated irreversibly in a distinct dimer configuration. Unexpectedly, the divalent cation Ca2? was not necessary for dimerization, although it greatly stabilized the protein-protein complex.     

Structure-based analysis reveals hydration changes induced by arginine hydrochloride

Arginine hydrochloride has been used to suppress protein aggregation during refolding and in various other applications. We investigated the structure of hen egg-white lysozyme (HEL) and solvent molecules in arginine hydrochloride solution by X-ray crystallography. Neither the backbone nor side-chain structure of HEL was altered by the presence of arginine hydrochloride. In addition, no stably bound arginine molecules were observed. The number of hydration water molecules, however, changed with the arginine hydrochloride concentration. We suggest that arginine hydrochloride suppresses protein aggregation by altering the hydration structure and the transient binding of arginine molecules that could not be observed.     

Mutated SEA-D227A-conjugated antibodies greatly enhance antitumor activity against MUC1-expressing bile duct carcinoma

For the purpose of establishing a new adoptive immunotherapy for bile duct carcinoma (BDC), we have directed our attention to superantigens (SAgs), the most potent known activators of T lymphocytes. In our previous study, staphylococcal enterotoxin A (SEA) was conjugated chemically with MUSE11 mAb, which recognizes the MUC1 cancer-associated antigen, and shown to enhance the specific cytotoxic activity of T-LAK cells against MUC1-expressing BDC cells (TFK-1) in vitro and in vivo. However, it is probable that SEA might cause side-effects because of nonspecific binding to class II positive cells. In order to overcome these, we generated mutated SEA (mSEA) by changing Asp at position 227 of native SEA to Ala, which has reduced affinity to MHC class II molecules, but retains the potential for T cell activation. When mSEA-D227A was administered to rabbits to examine effects on blood pressure, 500 times more mSEA-D227A was tolerated than native SEA. This prompted us to construct a mSEA-D227A-conjugated mAb, reactive with MUC1. It augmented the antitumor activity of T-LAK cells significantly, and furthermore, mSEA-D227A could be conjugated to two bispecific antibodies, BsAb (anti-MUC1 x anti-CD3) and BsAb (anti-MUC1 x anti-CD28), which in combination had greater enhancing effects than mSEA-D227A-conjugated anti-MUC1 mAb, and combination of unconjugated BsAbs. These findings indicate a utility of mSEA-D227A-conjugated antibodies for targeted cancer immunotherapy.     

A combination of 19F NMR and surface plasmon resonance for site-specific hit selection and validation of fragment molecules that bind to the ATP-binding site of a kinase

¹⁹F NMR has recently emerged as an efficient, sensitive tool for analyzing protein binding to small molecules, and surface plasmon resonance (SPR) is also a popular tool for this purpose. Herein a combination of ¹⁹F NMR and SPR was used to find novel binders to the ATP-binding pocket of MAP kinase extracellular regulated kinase 2 (ERK2) by fragment screening with an original fluorinated-fragment library. The ¹⁹F NMR screening yielded a high primary hit rate of binders to the ERK2 ATP-binding pocket compared with the rate for the SPR screening. Hit compounds were evaluated and categorized according to their ability to bind to different binding sites in the ATP-binding pocket. The binding manner was characterized by using isothermal titration calorimetry and docking simulation. Combining ¹⁹F NMR with other biophysical methods allows the identification of multiple types of hit compounds, thereby increasing opportunities for drug design using preferred fragments.     

Homogeneous noncompetitive immunoassay based on the energy transfer between fluorolabeled antibody variable domains (open sandwich fluoroimmunoassay).

The antigen-dependent stabilization of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 variable region was monitored with fluorescence resonance energy transfer (FRET) between fluorolabeled heavy chain (VH) and light chain (VL) fragments. The VH and VL fragments labeled with succinimide esters of fluorescein and rhodamine-X, respectively, were mixed in a cooled cuvette, and the change in fluorescence spectra upon antigen addition was monitored. When excited at 490 nm, significant decrease in the fluorescence at 520 nm and its increase at 605 nm were observed when an increasing amount of HEL was added to the mixture in the concentration range of 1-100 micrograms/mL. The assay, named open sandwich fluoroimmunoassay (FIA), is noncompetitive and homogeneous and can be conducted with one clone of antibody. With the use of appropriate antibodies, it is thought to be a quick and inexpensive alternative to the conventional laborious and/or expensive immunoassays.     

Structural and thermodynamical insights into the binding and inhibition of FIH-1 by the N-terminal disordered region of Mint3

Mint3 is known to enhance aerobic ATP production, known as the Warburg effect, by binding to FIH-1. Since this effect is considered to be beneficial for cancer cells, the interaction is a promising target for cancer therapy. However, previous research has suggested that the interacting region of Mint3 with FIH-1 is intrinsically disordered, which makes investigation of this interaction challenging. Therefore, we adopted thermodynamic and structural studies in solution to clarify the structural and thermodynamical changes of Mint3 binding to FIH-1. First, using a combination of circular dichroism, nuclear magnetic resonance, and hydrogen/deuterium exchange–mass spectrometry (HDX-MS), we confirmed that the N-terminal half, which is the interacting part of Mint3, is mostly disordered. Next, we revealed a large enthalpy and entropy change in the interaction of Mint3 using isothermal titration calorimetry (ITC). The profile is consistent with the model that the flexibility of disordered Mint3 is drastically reduced upon binding to FIH-1. Moreover, we performed a series of ITC experiments with several types of truncated Mint3s, an effective approach since the interacting part of Mint3 is disordered, and identified amino acids 78 to 88 as a novel core site for binding to FIH-1. The truncation study of Mint3 also revealed the thermodynamic contribution of each part of Mint3 to the interaction with FIH-1, where the core sites contribute to the affinity (ΔG), while other sites only affect enthalpy (ΔH), by forming noncovalent bonds. This insight can serve as a foothold for further investigation of intrinsically disordered regions (IDRs) and drug development for cancer therapy.     

Systematic approaches to using the FOX hunting system to identify useful rice genes

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23 000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis . This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.