Yoshihiro Kobashigawa

Role of the mobility of antigen binding site in high affinity antibody elucidated by surface plasmon resonance

Antibody is known to exhibit conformational change in the antigen recognition site after forming the initial complex. This structural change, which is widely known as “induced fit”, is believed to be critical for high affinity (Kd of nM range) of antigen-antibody interaction. Elucidation of this ‘induced fit’ process is essential for rational design of high affinity antibody, while it is prevented by limitation of the available biophysical and biochemical data of the initial complex. Here, we performed kinetic and thermodynamic analysis of the interaction between single-chain variable fragment (denoted as scFv) of 64M5 antibody and a (6-4) photoproduct by using surface plasmon resonance (denoted as SPR). It revealed that the 64M5scFv associates the (6-4) photoproduct at initial step by hydrophobic interactions, and enthalpy-driving interactions, hydrogen bonds and van der Waals interactions, were formed by second step structural rearrangement. Furthermore, mutational analysis revealed that the mobility of the antigen-binding site is critical for the second step. It could be assumed that optimization of the mobility of the antigen recognition site is a clue for rational design of high affinity antibody.

Nuclear magnetic resonance analysis of the conformational state of cancer mutant of fibroblast growth factor receptor 1 tyrosine kinase domain

Tyrosine kinases are key enzymes that play critical roles in growth signaling, the abnormal activation of which is associated with various human cancers. Activation of tyrosine kinases is mediated by tyrosine phosphorylation in the activation‐loop, which transforms the catalytic domain to the active state conformation. Cancer mutations are supposed to transform the conformation of the catalytic domain into the active‐form independent of the phosphorylation state of the activation‐loop. Here, we report structural and biophysical analyses of cancer mutations of the tyrosine kinase domain of fibroblast growth factor receptor 1 (FGFR1). Based on the nuclear magnetic resonance analyses, phosphorylation of the activation‐loop exhibited cooperative structural transition in the activation‐loop, C‐helix and P‐loop regions, whereas cancer mutations induced structural transformation at either one or two of these regions.

SIRT7 has a critical role in bone formation by regulating lysine acylation of SP7/Osterix

SP7/Osterix (OSX) is a master regulatory transcription factor that activates a variety of genes during differentiation of osteoblasts. However, the influence of post-translational modifications on the regulation of its transactivation activity is largely unknown. Here, we report that sirtuins, which are NAD(+)-dependent deacylases, regulate lysine deacylation-mediated transactivation of OSX. Germline Sirt7 knockout mice develop severe osteopenia characterized by decreased bone formation and an increase of osteoclasts. Similarly, osteoblast-specific Sirt7 knockout mice showed attenuated bone formation. Interaction of SIRT7 with OSX leads to the activation of transactivation by OSX without altering its protein expression. Deacylation of lysine (K) 368 in the C-terminal region of OSX by SIRT7 promote its N-terminal transactivation activity. In addition, SIRT7-mediated deacylation of K368 also facilitates depropionylation of OSX by SIRT1, thereby increasing OSX transactivation activity. In conclusion, our findings suggest that SIRT7 has a critical role in bone formation by regulating acylation of OSX.SP7/Osterix is a transcription factor involved in osteoblast differentiation. Here, the authors show that Sirtuin 7 activates Osterix posttranslationally by regulating its lysine acylation, and that mice lacking Sirtuin 7 in osteoblasts show reduced bone formation.

Design and tuning of a cell-penetrating albumin derivative as a versatile nanovehicle for intracellular drug delivery

&NA; Human serum albumin (HSA) is a superior carrier for delivering extracellular drugs. However, the development of a cell‐penetrating HSA remains a great challenge due to its low membrane permeability. We report herein on the design of a series of palmitoyl‐poly‐arginine peptides (CPPs) and an evaluation of their cell‐penetrating effects after forming a complex with HSA for use in intracellular drug delivery. The palmitoyl CPPs forms a stable complex with HSA by anchoring itself to the high affinity palmitate binding sites of HSA. Among the CPPs evaluated, a cyclic polypeptide composed of D‐dodecaarginines, palmitoyl‐cyclic‐(D‐Arg)12 was the most effective for facilitating the cellular uptake of HSA by HeLa cells. Such a superior cell‐penetrating capability is primarily mediated by macropinocytosis. The effect of the CPP on pharmacological activity was examined using three drugs loaded in HSA via three different methods: a) an HSA‐paclitaxel complex, b) an HSA‐doxorubicin covalent conjugate and c) an HSA‐thioredoxin fusion protein. The results showed that cell‐penetrating efficiency was increased with a corresponding and significant enhancement in pharmacological activity. In conclusion, palmitoyl‐cyclic‐(D‐Arg)12/HSA is a versatile cell‐penetrating drug delivery system with great potential for use as a nano‐carrier for a wide diversity of pharmaceutical applications.

Biophysical characterization of drug‐resistant mutants of fibroblast growth factor receptor 1

Over‐expression and aberrant activation of tyrosine kinases occur frequently in human cancers. Various tyrosine kinase inhibitors (TKIs) are under clinical use, but acquisition of resistance to these drugs is a major problem. Here, we studied the interaction between two drug‐resistant mutants of fibroblast growth factor receptor 1 (FGFR1), N546K and V561M, and four ATP‐competitive inhibitors, ponatinib, dovitinib, PD173074 and BGJ‐398. Among these protein–drug systems, the only marked reduction in affinity was that of PD173074 for the V561M mutant. We also examined the interaction of these FGFR1 variants to AMP‐PNP, a nonhydrolyzable analogue of ATP, and showed that N546K showed increased affinity for the ATP analogue as compared with the wild type. These findings will help to clarify the mechanism of drug resistance in mutant tyrosine kinases.