ドメイン蛋白質のコンホメーション安定性とコロイド安定性解析による抗体凝集機構の解明

学位の種別: 課程博士審査委員会委員 : （主査）東京大学客員教授 本田 真也, 東京大学教授 上田 卓也, 東京大学教授 伊藤 耕一, 東京大学准教授 深井 周也, 東京大学准教授 新井 宗仁University of Tokyo(東京大学

Establishment and Validation of Computational Model for MT1-MMP Dependent ECM Degradation and Intervention Strategies

MT1-MMP is a potent invasion-promoting membrane protease employed by aggressive cancer cells. MT1-MMP localizes preferentially at membrane protrusions called invadopodia where it plays a central role in degradation of the surrounding extracellular matrix (ECM). Previous reports suggested a role for a continuous supply of MT1-MMP in ECM degradation. However, the turnover rate of MT1-MMP and the extent to which the turnover contributes to the ECM degradation at invadopodia have not been clarified. To approach this problem, we first performed FRAP (Fluorescence Recovery after Photobleaching) experiments with fluorescence-tagged MT1-MMP focusing on a single invadopodium and found very rapid recovery in FRAP signals, approximated by double-exponential plots with time constants of 26 s and 259 s. The recovery depended primarily on vesicle transport, but negligibly on lateral diffusion. Next we constructed a computational model employing the observed kinetics of the FRAP experiments. The simulations successfully reproduced our FRAP experiments. Next we inhibited the vesicle transport both experimentally, and in simulation. Addition of drugs inhibiting vesicle transport blocked ECM degradation experimentally, and the simulation showed no appreciable ECM degradation under conditions inhibiting vesicle transport. In addition, the degree of the reduction in ECM degradation depended on the degree of the reduction in the MT1-MMP turnover. Thus, our experiments and simulations have established the role of the rapid turnover of MT1-MMP in ECM degradation at invadopodia. Furthermore, our simulations suggested synergetic contributions of proteolytic activity and the MT1-MMP turnover to ECM degradation because there was a nonlinear and marked reduction in ECM degradation if both factors were reduced simultaneously. Thus our computational model provides a new in silico tool to design and evaluate intervention strategies in cancer cell invasion

Conformational and Colloidal Stabilities of Human Immunoglobulin G Fc and Its Cyclized Variant: Independent and Compensatory Participation of Domains in Aggregation of Multidomain Proteins

Monoclonal immunoglobulin G (IgG) is a multidomain protein. It has been reported that the conformational and colloidal stabilities of each domain are different, and it is predicted that limited domains participate in IgG aggregation. In contrast, the influence of interdomain interactions on IgG aggregation remains unclear. The fragment crystallizable (Fc) region is also a multidomain protein consisting of two sets of C_H2 and C_H3 domains. Here, we have analyzed the conformational change and aggregate size of an aglycosylated Fc region induced by both acid and salt stresses and have elucidated the influence of interdomain interactions between C_H2 and C_H3 domains on the conformational and colloidal stabilities of the aglycosylated Fc region. Singular value decomposition analyses demonstrated that the C_H2 and C_H3 domains unfolded almost independently from each other in the aglycosylated Fc region. Meanwhile, the colloidal stabilities of the C_H2 and C_H3 domains affect the aggregation process of the unfolded aglycosylated Fc region in a compensatory way. Moreover, the influence of an additional interdomain disulfide bond, introduced at the C-terminal end of the C_H3 domains to produce the Fc variant, cyclized Fc, was evaluated. This interdomain disulfide bond increased the conformational stability of the C_H3 domain. The stabilization of the C_H3 domain in the cyclized Fc successfully improved aggregation tolerance following acid stress, although the sizes of aggregates produced were comparable to those of the aglycosylated Fc region

Conformational and Colloidal Stabilities of Isolated Constant Domains of Human Immunoglobulin G and Their Impact on Antibody Aggregation under Acidic Conditions

Antibody therapeutics are now in widespread use and provide a new approach for treating serious diseases such as rheumatic diseases and cancer. Monoclonal antibodies used as therapeutic agents must be of high quality, and their safety must be guaranteed. Aggregated antibody is a degradation product that may be generated during the manufacturing process. To maintain the high quality and safety of antibody therapeutics, it is necessary to understand the mechanism of aggregation and to develop technologies to strictly control aggregate formation. Here, we extensively investigated the conformational and colloidal characteristics of isolated antibody constant domains, and provided insights into the molecular mechanism of antibody aggregation. Isolated domains (CH2, CH3, CL, and CH1-CL dimer) of human immunoglobulin G were synthesized, solubilized using 49 sets of solution conditions (pH 2–8 and 0–300 mM NaCl), and characterized using circular dichroism, intrinsic tryptophan fluorescence, and dynamic light scattering. Salt-induced conformational changes and oligomer formation were kinetically analyzed by NaCl-jump measurements (from 0 to 300 mM at pH 3). Phase diagrams revealed that the domains have different conformational and colloidal stabilities. The unfolded fractions of CH3 and CH2 at pH 3 were larger than that of CL and CH1-CL dimer. The secondary and tertiary structures and particle sizes of CH3 and CH2 showed that, in non-native states, these domains were sensitive to salt concentration. Kinetic analyses suggest that oligomer formation by CH3 and CH2 proceeds through partially refolded conformations. The colloidal stability of CH3 in non-native states is the lowest of the four domains under the conditions tested. We propose that the impact of IgG constant domains on aggregation follows the order CH3 > CH2 > CH1-CL dimer > CL; furthermore, we suggest that CH3 plays the most critical role in driving intact antibody aggregation under acidic conditions

A Novel Protein Associated with Membrane-type 1 Matrix Metalloproteinase Binds p27kip1 and Regulates RhoA Activation, Actin Remodeling, and Matrigel Invasion*

Pericellular proteolysis by membrane-type 1 matrix metalloproteinase (MT1-MMP) plays a pivotal role in tumor cell invasion. Localization of MT1-MMP at the invasion front of cells, e.g. on lamellipodia and invadopodia, has to be regulated in coordination with reorganization of the actin cytoskeleton. However, little is known about how such invasion-related actin structures are regulated at the sites where MT1-MMP localizes. During analysis of MT1-MMP-associated proteins, we identified a heretofore uncharacterized protein. This protein, which we call p27RF-Rho, enhances activation of RhoA by releasing it from inhibition by p27kip1 and thereby regulates actin structures. p27kip1 is a well known cell cycle regulator in the nucleus. In contrast, cytoplasmic p27kip1 has been demonstrated to bind GDP-RhoA and inhibit GDP-GTP exchange mediated by guanine nucleotide exchange factors. p27RF-Rho binds p27kip1 and prevents p27kip1 from binding to RhoA, thereby freeing the latter for activation. Knockdown of p27RF-Rho expression renders cells resistant to RhoA activation stimuli, whereas overexpression of p27RF-Rho sensitizes cells to such stimulation. p27RF-Rho exhibits a punctate distribution in invasive human tumor cell lines. Stimulation of the cells with lysophosphatidic acid induces activation of RhoA and induces the formation of punctate actin structures within foci of p27RF-Rho localization. Some of the punctate actin structures co-localize with MT1-MMP and cortactin. Down-regulation of p27RF-Rho prevents both redistribution of actin into the punctate structures and tumor cell invasion. Thus, p27RF-Rho is a new potential target for cancer therapy development