Studies on the functional role of HVR domain in the small Gprotein H-Ras and its application to Artificial control of Ras

• 資料種別: 博士論文
• タイトル: Studies on the functional role of HVR domain in the small Gprotein H-Ras and its application to Artificial control of Ras
• 著者・編者: Nahar, Rufiat; Rufiat, Nahar
• 著者標目: Nahar, Rufiat; Rufiat, Nahar
• 出版年月日等: 2022-03-18
• 出版年（W3CDTF）: 2022-03-18
• 授与機関名: 創価大学
• 授与年月日: 2022-03-18
• 授与年月日（W3CDTF）: 2022-03-18
• 報告番号: 甲第190号
• 学位: 博士（工学）
• 本文の言語コード: eng
• 件名標目: Small G protein; H-Ras; caged compound; photochromic molecule; chemical modification
• 対象利用者: 一般
• 一般注記: Ras is one of the Small G proteins which is known as a molecular switch, is a central regulator of cellular signal transduction processes leading to transcription, cell cycle progression etc. The active ON state and the inactive OFF state of Ras are regulated by the two factors. GTPase activating protein (GAP) induce hydrolysis of GTP to GDP resulting in formation of inactive state of Ras. On the other hand, Guanine nucleotide exchange factor (GEF) replaces GDP with GTP and make Ras ON state. The switching mechanisms utilizing conformational changes in the nucleotide-binding motifs have been well studied at the molecular level. Interestingly, recent studies have shown that G proteins have a common nucleotide-binding motif with the ATP-driven motors, myosin and kinesin. These nucleotide binding proteins might be evolved from a common nucleotide-binding ancestral protein and share a common catalytic core region including switch I, Switch II and P-loop, and molecular mechanism utilizing a nucleotide hydrolysis cycle . Previously Studies demonstrated that incorporation artificial regulatory nanodevices such as a photochromic molecule into the functional site of Kinesin enable to control ATPase activity photoreversibly . Interestingly, Hyper Variable Region (HVR) is one of the functional parts of the G proteins in which modification induce multimerization and interaction with plasma membrane of Ras. In physiological state Ras forms the nanocluster on the plasma membrane by the lipid modifications of the hypervariable region (HVR) at C-terminal to exhibit physiological function . It is believed that this HVR domain might play a crucial role in Ras protein to its cellular function. First in this study, we have demonstrated that chemical modification of cysteines residues in HVR with caged compounds instead of lipidation induces multimerization of H-Ras. Sulfhydryl-reactive caged compound, 2-Nitrobenzyl bromide (NBB) was stoichiometrically incorporated into the cysteine residue of HVR and induced formation of Ras multimer. Light irradiation induced elimination of 2-Nitrobenzyl group, resulting in conversion of multimer to monomer. SEC-HPLC and Small angle X-ray scattering (SAXS) analysis revealed that the H-Ras forms pentamer. Electron microscopic observation of the multimer showed circular ring shape which is consistent with the structure estimated from X-ray scattering. The shape of the multimer may reflect the physiological structural state of Ras. It was suggested that the multimerization and monomerization of H-Ras was controlled by the modification with caged compound at HVR and light irradiation reversibly. However, caged compound exhibit irreversible photo eliminating reaction. Therefore, caged compounds does not work as a reversible photo-switch. In further study, we employed the azobenzene derivative as a photo reversible nano switching device and incorporated into the HVR to control Ras function. We introduced the two highly different polarity photochromic sulfhydryl-reactive azobenzene derivatives, 4-phenylazophenyl maleimide (PAM) and 4-chloroacetoamido-4’-sulfo-azobenzene (CASAB) into cysteine residues in HVR to regulate the GTPase activity by photoirradiation. PAM was stoichiometrically incorporated into the three cysteine residues in HVR and induced multimerization. The PAM-modified mutants exhibited reversible alterations in GTPase activity accelerated by GEF And GAP, and multimerization accompanied by photoisomerization upon exposure to ultraviolet and visible light irradiation. CASAB was incorporated into two of the three cysteine residues in HVR but not induced multimerization. GTPase of the H-Ras modified with CASAB was photocontrolled more effectively than PAM-H-Ras. Interestingly CASAB modification did not induced H-Ras multimerization. The results suggest that incorporation of photochromic molecules into its functional site enables photoreversible control of the function of the small G protein Ras. Well known photochromic compounds show light sensitivity at a specific wavelength. Upon light irradiation photochromic compounds can change their structure and functions. There are two types of mechanisms observed for returning to their original states. A mechanism that returns by irradiating light with a different wavelength, it’s called P-type such as diarylethene and fulgide. Another one, a mechanism that returns by heat, it’s called T-type such as spiropyran, azobenzene, and stilbenes. In this study we used small caged compound which may mimic the physiological lipidation and photochromic compound such as azobenzene derivatives. We employed a sulfhydryl group reactive caged compound to modify the cysteine residues in HVR. NBB used in this study is one of the well-known cage compounds and can be specifically introduced into the thiol group of the cysteine residue as shown in Then, by light irradiation at 340-400 nm, the nitrobenzyl group is eliminated and the protein reversibly returns to its original state. There are 6 cysteine residues in Human H-Ras. Three of them are in the globular domain and C118 is located on the surface. The remaining three cysteine (C181, C184,C186) which are known as lipidation sites are in the HVR domain. HVR domain is exposed to solvent. NBB is incorporated in to the cysteine residues stoichiometrically. Therefore, it is assumed that the four cysteine residues (C118, C181, C184 and C186) exposed to solvent are modified specifically.
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• コレクション（共通）: 障害者向け資料
• コレクション（障害者向け資料：レベル1）: テキストデータ
• コレクション（個別）: 国立国会図書館デジタルコレクション > デジタル化資料 > 博士論文; https://dl.ndl.go.jp/collections/A00014
• 収集根拠: 博士論文（自動収集）
• 受理日（W3CDTF）: 2023-10-11T15:41:03+09:00
• 記録形式（IMT）: application/pdf
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• デジタル化資料送信: 図書館・個人送信対象外
• 遠隔複写可否（NDL）: 可
• 掲載誌（URI）: http://hdl.handle.net/10911/00040741; https://soka.repo.nii.ac.jp/records/40748
• 連携機関・データベース: 国立国会図書館 : 国立国会図書館デジタルコレクション; https://dl.ndl.go.jp