行政院國家科學委員會補助專題研究計畫成果報告 Transglutaminase 於培養細胞對高溫及氧化壓力反應所扮演之角色計畫類別 :X 個別型計畫整合型計畫計畫編號 :: NSC B 執行期間 : 95 年 08 月 01 日至 96 年 07 月 31 日

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2 行政院國家科學委員會補助專題研究計畫成果報告 Transglutaminase 於培養細胞對高溫及氧化壓力反應所扮演之角色計畫類別 :X 個別型計畫整合型計畫計畫編號 :: NSC B 執行期間 : 95 年 08 月 01 日至 96 年 07 月 31 日計畫主持人 : 張震東共同主持人 : 本成果報告包括以下應繳交之附件 : 赴國外出差或研習心得報告一份赴大陸地區出差或研習心得報告一份出席國際學術會議心得報告及發表之論文各一份國際合作研究計畫國外研究報告書一份執行單位 : 國立台灣大學生命科學院生化科學研究所中華民國 96 年 10 月 22 日 1

3 行政院國家科學委員會專題研究計畫成果報告國科會專題研究計畫成果報告撰寫格式說明 Preparation of NSC Project Reports 計畫編號 :NSC B 執行期限 :95 年 8 月 1 日至 96 年 7 月 31 日主持人 : 張震東國立台灣大學生命科學院生化科學研究所一中文摘要轉穀胺醯氨酶摧化之反應基本上是以酵素 Cys 的 thiol group 去攻打 Gln 上的 carbonyl group,nh3 離開, 形成 Thioester bond 而此 Thioester bond 大多是接受 Lys 的 ε-amino group 攻擊形成 Isopeptide bond 而酵素離開此 Isopeptide bond 亦可接受另一個一級氨水分子或者醇類的攻擊因此此酵素作用的反應為 Transadmidation 造成蛋白質之聚合 Amine incorporation 造成 polyamines 共價結合上蛋白質 Esterification 造成 Gln 轉換成酸酯 Deamidation 造成 Gln 轉換成 Glu 及 Isopeptide cleavage 造成聚合蛋白質的分開其主要功能在於凝血瘍口癒口調控細胞自戕死亡神導傳導物質釋放訊息傳導及細胞間質形成等轉穀胺醯氨酶之功能必需透過受質之鑑定以及 Transamidation 造成之影響オ得以瞭解本實驗室以新穎方法來純化及鑑定轉穀胺醯氨酶之受質, 小鼠 (>10 周齡 ) 之肝臟及睪丸其轉穀胺醯氨酶酵素活性, 並純化出受質, 通過質譜分析鑑定受質的身分, 最後以免疫轉漬法以確認受質之真正身分在超過一白個鑑定受質中, 大概屬下列性質蛋白 ; 細胞骨架及其調節蛋白 Chaperones 及 Co-chaperones 內質網蛋白細胞 Detoxification 用蛋白蛋白質轉譯調節蛋白等, 大多是與細胞壓カ反應有關之蛋白更重要的是其中多個受質屬新的發現由於轉穀胺醯氨酶本身是氧化壓力所活化的酵素, 而且多數受質屬壓力反應有關之蛋白, 因此我們認為轉穀胺醯氨酶及其受質参與細胞壓力反應, 其扮演之角色則是本計畫探討的主題關鍵詞 : 轉穀胺醯氨酶質譜分析受質 Abstract Transglutaminases (TG) are Ca 2+ -dependent enzymes which catalyze a post-translational modification of proteins. The enzyme reaction leads to the formation of an isopeptide bond either within or between polypeptide chains. The γ-glutamyl-ε-lysine crosslinks are formed between the γ-carboxamide group of peptide-bound glutamine residues and the ε-amino group of peptide-bound lysine residues. Polyamines can replace lysine residue in the transamidation reaction in vitro and in vivo. Transglutaminases are abundant enzymes which are involved in a number of different physiologic processes, e.g., plasma transglutaminase factor XIII stabilizes the fibrin clot during hemostasis, and keratinocyte and epidermal transglutaminase contribute to the formation of the cornified envelope in skin. Several physiological roles for tissue transglutaminases have been demonstrated, such as wound healing, fibrosis, apoptosis, and matrix formation. In order to understand the physiological functions of tissue transglutaminase (ttg or TG2), one needs to identify the acyl donor and the acyl acceptor substrates in the transamidation reaction. To this end, we have identified 29 potential transglutaminase substrates from mouse liver extract by substrate purification and tandem Mass Spectrometry analysis. Interestingly, chaperones and co-chaperones are the most abundant ttg substrates found. Other transglutaminase substrates include intermediate filament protein, β-actin, proteasome proteins, peroxiredoxin, proteins, valosin-containing protein, nucleolin, glyoxalase 1. Many of the ttg substrates are cellular proteins with functions related to cellular response to stress. More importantly, some of the ttg substrates have not been reported. In light of the fact that oxidative stress or UV irradiation elevates in situ ttg activity, we seek to study the role of ttg in HepG2 cells in response to heat shock and oxidative stress. More specifically, we will determine whether ttg is up-regulated or activated in HepG2 cells in response to heat shock and oxidative stress and further to determine the consequences of ttg activation. In addition, we will examine whether the ttg substrates that we have identified in mouse liver extract are indeed modified in vivo in HepG2 cells. In the end, we wish to know the roles of ttg and heat shock proteins and their interplay in HepG2 cells in response to heat shock and oxidative stress. Keywords: transglutaminase substrates proteomics Mass spectrometry 二緣由與目的轉穀胺醯氨酶家族有三組成員 :Papain-like transglutaminases protein disulfide isomerase-like transglutaminases 及 bacterial toxin transglutaminases Papain-like transglutaminases 屬於 Cysteine protease 2

4 superfamily 的一員, 其它次家族成員有 papain calpain foot and mouth virus protease deubiquitylating enzymes 及 N-acetyltransferases (Laszlo and Graham, 2003) 這些酵素具有與摧化反應相關的結構如 Cys-His-Asn 或 Cys-His-Glu, 稱 catalytic triad 人類全基因含九個 transglutaminase 基因, 其中有一個是 erythrocyte band4.2 巳不具摧化能力, 其詳細特性如表一所列巳知的蛋白質均無醣化修飾, 也缺乏雙硫鍵, 摧化機制需要鈣離子 (mm) 的存在 Type 2 transglutaminase(tg2) 是第一個被發現的成員, 幾乎表現在所有的器官, 也表現在細胞的不同部位如 cytosol (80%) plasma membrane ( 含 extracellular matrix,10-15%) nuclear membrane (5%) 雖然一級結構缺乏 Signal peptide,tg2 卻可以分泌到細胞表面及細胞間質上最奇特的是它又是個 GTP-binding protein, 結合 GTP 會抑制 transglutaminase 酵素活性, 但是它又具 GTPase 活性,GTP 終究水解為 GDP 不過若 TG2 先結合了鈣離子, 鈣離子可阻斷 GTP 的結合因此 TG2 與高分子量 G protein (Ghα) 是同一個蛋白質, 可媒介 αadrenergic receptor 活化所造成之 phospholipase C 的活化 (Nakaoka et al., 1994) 轉穀胺醯氨酶之受質鑑定有一種較傳統的方法, 是把純化蛋白加入酵素看蛋白質是否聚合, 或再加入 5-(biotinamido)pentylamine 為探針去標示蛋白質, 不過這種作法之生理意義較受質疑, 實驗靠內生性酵素作反應較有意義轉穀胺醯氨酶之受質鑑定有二個對象 ; 一稱 Acyl donor 指提供 Gln 參與反應的蛋白質, 另一稱 Acyl acceptor 指提供 Lys 參與反應的蛋白質, 此二對象一般是二個不同之蛋白質亦可以是同一個蛋白質, 形成 Intramolecular isopeptide bond, 或是 Heterodimer 所以在受質鑑定就有二群對象 ; 以 5-(biotinamido)pentylamine 為探針 ( 或 3 [H]-putrescine), 經摧化反應後可標示到 Acyl donor, 此標示之蛋白質可轉漬至 PVDF 後以 Streptavidin-peroxidase 呈色 ( 或以 X 光片 ) 顯示, 或經 Streptavidin-agarose 親和力管柱純化至於 Acyl acceptor 之鑑定文獻上則有不同的 biotinylated peptide 的設計, 其中序列中含 Gln 氨基酸 ; 如 LGLGQGKVLG (Gorman and Folk, 1984) TVQQEL (Ruoppolo et al., 2003) GQQQLG (Hu and Messersmith, 2003) QQIV (Lorand et al., 1992) 等 Pastor 等 (1999) 曾對此設計作過詳細探討, 結論如下 : 1. Gln 不可在 N- 或 C-terminus 2. Gln 不可位在二個鹼性胺基酸中間或 Pro 中間在文獻上報導的 Acyl donor 較多, 可能是因為 5-(biotinamido)pentylamine 及 3 [H]-putrescine 較易取得, 同時也不必作設計針對這問題, 作者巳設計合成了一個新的探計 Biotin-RSGQQQLGSS, 反應極佳而且水溶解度非常好, 因此本計畫將以此胜肽及 5-(biotinamido)pentylamine 為探針, 尋找及鑑定轉穀胺醯氨酶之受質在本計畫中, 我們利用小鼠的睪丸為材料, 以蛋白質體學的方法去純化以及鑑定小鼠睪丸內 TG 的受質, 希望在一個像 testis 這樣持續進行細胞生長與分化的組織裡, 了解 TG 所扮演的角色為何經由純化及 MS/MS 鑑定過後, 我們發現了數十種蛋白質在小鼠睪丸內可能為 TG 的受質, 在經由重組 TG 受質蛋白的 in vitro transamidation 反應以及 TG substrates pull-down 的 immunoblotting assay, 我們也證實了這些蛋白在 in vitro 的確為 TG 的受質未來我們希望從中選取了幾個可能為 TG 受質的蛋白, 觀察其 TG 進行 transamidation 過後, 會對這些蛋白的酵素活性 protein-protein interaction 或是 protein translocation 會有怎樣的影響, 進而能更加了解 TG 在 testis 內的角色與功能三材料與方法蛋白質膠體電泳電泳配方及條件參考 Schägger & Jagow (1987) 所著 Tricine-SDS 方法,Running gel 之濃度為 7.5%, stacking gel 濃度為 4%,Cathode buffer 組成是 0.1 M Tris 0.1M Tricine 0.1% SDS,pH 8.25 Anode buffer 組成是 0.2 M Tris, ph 8.9 Gel buffer 組成是 1 M Tris 0.1% SDS,pH 8.45 電泳條件是五十伏特二十分鐘及一百伏特七十分鐘之後膠體以水洗六十分鐘後以 Colloidal Coomassie blue G-250 染色六十分鐘, 再以水洗淨二十分鐘銀染色則採取 Amersham-Pharmacia Plus One Silver Stain Kit,並依原廠商提供步驟進行染色,但是退染改為 5% 醋酸. 近免疫轉漬法 (Near-immunoblotting) 將欲分析之材料進行膠體電泳分析, 利用 Diffusion blotting (Chen and Chang, 2001) 將蛋白質轉印至 PVDF 濾紙上以 Phosphate buffered saline (PBS) 洗滌十分鐘, 加入 Blocking solution (5% skim milk in PBS, 0.05% Tween 20) 室溫反應一小時, 再以 PBS 洗滌三次接著加入 Rabbit IgG-streptavidin complex (1 ug/ul 於 PBS, 含 0.05% Tween 20 3 mg/ml BSA) 於室溫反應一小時, 再以 PBS-T 洗滌三次, 然後加入 Peroxidae-conjugated anti-rabbit IgG 抗體 ( 溶於 PBS 含 0.05% Tween 3 mg/l BSA) 反應於室溫一小時, 再以 PBS 洗滌三次最後以 DAB (0.6 mg/ml) 0.01% 氯化鎳 1 ul/ml 雙氧水呈色轉穀胺醯氨酶摧化反應我們取成鼠 Testis 以 10 重量體積之 20 mm Tris, ph 8, and 2% Triton X-100 溶液研磨後, 以 20,000 xg 離心三十分鐘分成分上清液及不溶物二部分不溶物再以 20 mm Tris, ph8, and 0.15M NaCl (TBS) 清洗一次轉穀胺醯氨酶摧化反應含 0.2 ug/ul 5-(biotinamido)pentylamine (bpa) 或 biotin-rsgqqqlgss (bpq) 以及 5 mm CaCl 2, and 0.1 mm DTT 及適量之組織材料反應於室溫進行三十分鐘, 有時加入 20 mm of dansylcadaverine 當作抑制劑上清液以加入等體積之 2x SDS sample buffer 中止反應而不溶物靠加入 3

5 Urea 到 8M 濃度, 混合十分鐘後離心取得可溶蛋白轉穀胺醯氨酶受質純化因為要作純化所以上清液中止反應改成加入 Urea 到 8M DTT 到 50 mm, 於室溫一小時後加入 Iodoacetamide 到 100 mm, 再置於室溫一小時之後材料以 Amersham Biosciences 出產之 2-D clean-up kit 作蛋白質沈澱, 後溶於 8M urea/tbs 中, 與等體積之 Streptavidin-argarose beads ( 以 8 M urea/tbs 平衡 ) 結合, 後以五倍體積 8 M urea/1.0 M NaCl/TBS, 0.1% SDS/TBS, 及 20mM Tris buffer 清洗 Streptavidin-agarose beads 各一次, 最後以二倍體積 4% SDS 並加熱將受質沖洗出來西方墨點法 (Western blotting) 將欲分析之材料進行膠體電泳分析, 利用 Semi-dry blotter (Hoeffer Semi-Phor) 將蛋白質轉印至 PVDF 濾紙上以 Phosphate buffered saline (PBS) 洗滌十分鐘, 加入 Blocking solution (5% skim milk in PBS) 室溫反應一小時, 再以 PBS 洗滌三次接著加入抗血清 ( 一比一千稀釋於 PBS, 含 1 mm EDTA 3 mg/ml BSA) 於攝氏四度反應十六小時, 再以 PBS 洗滌三次, 然後加入 Peroxidase-conjugated anti-guinea pig IgG 抗體 ( 溶於 PBS 含 1 mm EDTA 3 mg/l BSA) 反應於室溫二小時, 再以 PBS 洗滌三次最後以 DAB (0.6 mg/ml) 0.01% 氯化鎳 1 ul/ml 雙氧水呈色質譜分析 (Mass spectrophotometry analysis and protein in-gel digestion) 將純化得到之蛋白質作 SDS 電泳, 以 Coomassie blue 染色, 待腿色後, 以刀片將色帶切下, 放入微量管中, 加入 100 µl 之 DTT/25 mm 碳酸氫氨於 37ºC 反應一小時, 加入 100 µl Acetonitrile, 振盪數分鐘, 離心除去上清液加入 100 µl 65 mm Iodoacetamide/25 mm 碳酸氫氨, 於室溫反應一小時, 加入 100 µl Acetonitrile, 振盪數分鐘, 離心除去上清液加入 200 µl 50% Acetonitrile/25 mm 碳酸氨, 振盪數分鐘, 離心除去上清液加入 200 µl 100% Acetonitrile, 振盪數分鐘, 離心除去上清液加入 µl trypsin 溶液 ( µg 配於 25 mm 碳酸氨 ), 於 37ºC 反應 16 小時, 加入 10 µl 100% Acetinitrile, 振盪數分鐘, 離心收集上清液殘餘膠體加入 20 µl 100% Acetonitrile/0.1% TFA 萃取, 振盪數分鐘, 離心收集上清液殘餘膠體加入 20 µl 50% Acetonitrile/0.1% TFA 萃取, 振盪數分鐘, 離心收集上清液殘餘膠體加入 20 µl 100% Acetonitrile/0.1% TFA 萃取, 振盪數分鐘, 離心收集上清液把各次收集之上清液混合, 並以 SpeedVac 抽至殘留體積約 10 µl, 送中央研究院基因體 / 蛋白質體中心, 以 LC-Mass-Mass 作蛋白質定序四結果與討論我們不將 mouse cytosol 直接進行 in vitro transamidation 的反應, 再利用 ammonium sulphate 做沉澱, 以 8 M urea/20 mm Tris-HCl 回溶沉澱後的蛋白, 並利用 streptavidin argerose 進行純化步驟最後純化到的 binding proteins, 經電泳及 Coomassie blue 染色解析後, 將 SDS-PAGE 上的 lane( 包含 bpq 和 bpa) 對分為二十等分, 操作 in-gel digestion, 將 tryptic peptides 由膠片中萃取出, 接著進行 MS/MS 的分析 ( 圖一 ), 鑑定出的蛋白身分詳列於表一經由蛋白質質譜鑑定, 最後鎖定 12 個我們有興趣研究的 TG substrates, 進行重組蛋白的製備所選用的表現載體為 pet21b, 使用 E. coli BL21 作為宿主, 表現我們所挑選出蛋白的部分片段預測這幾個蛋白的分子量理論值 :LDH testis isoform 為 36 kda NSFL1 cofactorp47 為 41 kda nudc 為 38 kda Peroxiredoxin-1 為 22 kda Retinal dehydrogenase 為 54 kda TOM34 為 34 kda Glutathione S-transferase Mu 5 為 27 kda HSP70 為 70 kda APG-1 為 94 kda Poly(A)-binding protein 1 為 71 kda Heterogeneous nuclear ribonucleoprotein K 為 51 kda hnrnp A2/B1 為 36 kda 在這裡我們盡量去表現出全長的蛋白, 但其中 HSP70 與 APG-1 僅表現出 1/3 與 1/2 的長度, 這些表現蛋白在經過 induction 以及純化之後, 接著進行 in vitro transamidation 以及 biotin overlay assay 由結果顯示 ( 圖二 ), 在有外加 TG 的條件下, 這些重組蛋白會有 bpa incorporation 的情形發生, 有些甚至會有高分子量的 complex 形成, 懷疑是由於 TG 的 cross-linking 作用而將其重組蛋白本身作了 cross-linking 所導致將純化到之 binding proteins, 進行 SDS-PAGE 及轉印後, 以各分子的抗體血清操作西方轉印法 ( 圖三 ) 由圖三結果我們可以發現,albumin, nudc, hsp70, tubulin, apg1 及 hsp90 在沒有加入任何的 biotin probe sample 裡, 看不到訊號的產生, 而在加入 bpq 或是 bpa 的 sampl 裡, 可以看到有蛋白存在的訊號在此次的 TG substrate 純化與鑑定中, 我們一共使用了兩種不同的 probes(bpa: 5-biotin-amidopentylamine bpq:biotin-sgqqqlgss ), 其中 bpa 作為一個 K donor, 提供 TG 反應時所需的 amino group,bpq 作為一個 Q donor, 提供 TG 反應時所需的 carboxamide group 所以本次實驗經由 MS/MS 的分析結果, 有些蛋白是 TG 利用 bpa 所鑑定到的受質, 有些則是 TG 利用 bpq 所鑑定到的受質, 代表這些蛋白有些是利用本身的帶有的 carboxamide group 與 bpa 反應, 有些則是利用本身的 amino group 與 bpq 進行反應而有些蛋白同時被鑑定為 bpa 和 bpq 都可反應的分子時, 代表這類的分子本身應該同時具有 Q donor 和 K donor, 而這樣的分子本身就容易被 TG 作用, 產生 oligomer 像是 Poly A-binding protein 1 Heat shock protein 70.2 Retinal dehydrogenase Glutathione S-transferase 1 Peroxiredoxin 4

6 等 TG substrates Molecula Function Mascot Fragments Accession # bpa in testis r size score found Serum albumin 67 kd Serum protein P07724 Alpha-2-macroglobulin 220 kd Protease inhibitor Q61838 Pyruvate carboxylase 130 kd Endogenous Z14044 biotin-conjugated protein Clathrin heavy chain 90 kd Endocytosis Q68FD5 (HLA-B-associated 83 kd Contains ubiquitin-like 87 1 Q6ZQ38 transcript 3) domain Poly(A)-binding protein1 71 kd Pre-mRNA splicing P29341 Heterogeneous nuclear 96 kd mrna processing, Q8VDM6 ribonucleoprotein U-like protein 1 transcription repressor Glial fibrillary acidic 50 kd Glial intermediate filament 61 5 P03995 protein, Tubulin alpha-6 chain 50 kd Microtubule component P68373 Heat shock 70 kda protein 94kD HSP 70 family chaperone Q Hexokinase kd Glycolysis enzyme 56 2 P17710 Protein KIAA kd Unknown 67 2 Q8VDP4 homolog Heat shock protein HSP 86 kd Molecular chaperone P alpha Heat shock protein HSP 86 kd Molecular chaperone P beta Piwi-like protein 1 98 kd RNA-binding protein Q9JMB7 Microtubule-associated 117 kd Promotes microtubule P27546 protein 4 assembly Polypyrimidine 75 kd Pre-mRNA splicing 73 2 Q8VIJ6 tract-binding protein-associated-splicin g factor) Sperm equatorial segment 45 kd Acrosome protein 67 3 Q9D5A0 protein 1 Heat shock-related 70 kda 70 kd Molecular chaperone P17156 protein 2 Dipeptidase 3 54 kd GPI-anchored dipeptidase 83 2 Q9DA79 5

7 TG substrates Molecula Function Mascot Fragments Accession # bpa in testis r size score found RNA-binding protein 68 kd Possible transcription 79 3 Q61545 EWS repressor L-amino acid oxidase 70 kd Lysosome enzyme 70 2 O09046 Matricin 70 kd TCP-1 gamma chaperone P80318 RNA-binding protein 53 kd Nuclear riboprotein 92 3 P56959 FUS Elongation factor Tu 50 kd Protein translation P10126 Alpha-2-antiplasmin 55 kd Serine protease inhibitor 84 4 Q61247 Propionyl-CoA 80 kd Endogenous 70 3 Q91ZA3 carboxylase alpha chain biotin-conjugated protein Retinal dehydrogenase 1 54 kd Aldehyde dehydrogenase P24549 UV excision repair 43 kd DNA repair P54728 protein RAD23 homolog B Heterogeneous nuclear 51 kd Pre-mRNA splicing P61979 ribonucleoprotein K Y-box-binding protein 2 38 kd Pre-mRNA splicing Q9Z2C8 T-complex protein 1 60 kd Molecular chaperone 73 3 P11984 subunit alpha A Nucleoporin-like protein 58 kd Acrosome biogenesis 68 3 Q8K2K6 RIP Alpha-tubulin 3/7 50 kd Microtubule component P05214 GPI-anchored protein 73 kd Unknown Q60865 p137 Drebrin-like protein 49 kd F-actin binding 60 2 Q62418 NSFL1 cofactor p47 41 kd Golgi fragmentation and Q9CZ44 reassembly ATP-dependent RNA 46 kd Protein translation P60843 helicase eif4a-1 Septin-2 (Protein 41 kd Cytokinesis 72 2 P42208 NEDD5) AU-rich element 38 kd mrna turnover 56 3 Q60668 RNA-binding protein 1 Beta-actin 42 kda Microfilament component P60710 hnrnp X 38 kda RNA binding Q

8 40S ribosomal protein SA 33 kda Ribosomal protein P14206 LDH testis isoform 36 kda Glucose metabolism P00342 APC-binding protein EB1 30 kda Microtubule and spindle Q61166 formaiton protein epsilon 29 kda Phosphoprotein binding P62259 TG substrates Molecula Function Mascot Fragments Accession # bpa in testis r size score found protein theta 28 kda Phosphoprotein binding P protein zeta/delta 28 kda Phosphoprotein binding P63101 Tubulin beta-2c 50 kda Microtubule component P68372 LDH-A 36 kda Glucose metabolism P06151 DEAD box RNA helicase 69 kda Pre-mRNA splicing Q61656 DEAD1 Peroxiredoxin-4 31 kda Redox regulation O08807 Glutathione S-transferase 27 kda Testis GST P48774 Mu 5 hnrnp-e1 37 kda RNA binding P60335 snrnp-b 24 kda Pre-mRNA splicing 83 2 P27048 hnrnp F 45 kda Pre-mRNA splicing 80 4 Q9Z2X1 Glutathione S-transferase 26 kda Cell detoxification P24472 A4 Peroxiredoxin-2 21 kda Redox regulation Q61171 hnrnp H 49 kda Pre-mRNA splicing O35737 TG substrates bpq in testis Molecula r size Function Mascot score Fragments found Accession # Serum albumin 67 kd Serum protein P07724 Pyruvate carboxylase 129 kd Endogenous Q05920 biotin-conjugated protein Alpha-tubulin 3/7 50 kd Microtubule component P05214 Clathrin heavy chain 191 kd Endocytosis 98 2 Q68FD5 MAP kd Microtubule assembly 65 2 P27546 Elongation factor Tu 50 kd Protein translation P10126 GRP94; HSP 90 beta 92 kd Molecular chaperone P08113 HSP 90 alpha 85 kd Molecular chaperone P07901 Hsp kd Mitochondria chaperone 96 2 Q9CQN1 Beta-actin 42 kd Microfilament component P60710 eif-4b 69 kd Protein translation Q8BGD9 7

9 Nucleolin 77 kd Nucleolar protein 100, 72 2, 2 P09405 Spermatid-specific 58 kd Spermatogenesis 89 2 Q6P902 thioredoxin-1 Transferrin 77 kd Fe (III) binding 63 2 Q921I1 GFAP 50 kd Glial intermediate filament 63 2 P03995 HSP kd Molecular chaperone P17156 Heat shock cognate kd Molecular chaperone P63017 kda protein Cortactin 61 kd Src substrate Q60598 Propionyl-CoA 80 kd Endogenous Q91ZA3 carboxylase alpha chain biotin-conjugated protein DEAD box RNA helicase 69 kd Pre-mRNA splicing Q61656 DEAD1 Heat shock 70 kda 94 kd Molecular chaperone 86 2 P48722 protein 4L induced by osmotic stress Kininogen-1 73 kd Cys protease inhibitor O08677 Y-box-binding protein 2 38 kd mrna processing 62, 95 2, 1 Q9Z2C8 Retinal dehydrogenase 1 54 kd Aldehyde dehydrogenase P24549 Tubulin beta-2c 49 kd Microtubule component P68372 ALDH class 2 56 kd Aldehyde dehydrogenase P47738 PAI mrna-binding 45 kd mrna stability 94 1 Q9CY58 protein 1 Unc-33-like 62 kd Synaptic vesicle protein 66 2 Q62188 phosphoprotein) hnrnp F 46 kd Pre-mRNA splicing Q9Z2X1 TG substrates Molecula Function Mascot Fragments Accession # bpq in testis r size score found Proacrosin-binding 61 kd Acrosome packaging Q3V140 protein sp32 Argininosuccinate 46 kd Urea cycle enzyme P16460 synthase hnrnp H 49 kd Pre-mRNA splicing 80 2 O35737 Testis-specific gene A2 34 kd Male miosis Q8VIG3 hnrnp A3 40 kd Pre-mRNA splicing Q8BG05 Septin-2 (NEDD5) 42 kd Cytokinesis 96 2 P42208 Leucine-rich repeat-containing protein kd Unknown 74 2 Q9DAP0 8

10 nudc 38 kd Mitotic spindle formation O35685 GAPDH 36 kd Glycolysis enzyme P16858 Suppressor of G2 allele of 38 kd SCF complex component Q9CX34 SKP1 homolog hnrnp A2/B1 36 kd Pre-mRNA splicing O88569 hnrnp C1 / C2 34 kd Pre-mRNA splicing 89 2 Q9Z204 TOM34 34 kd Mitochondria protein Q9CYG7 import Protein phosphatase 1C 37 kd Signal transduction 84 2 P63087 catalytic subunit LDH testis subunit 36 kd Glycolysis P00342 Swiprosin-1 27 kd Calcium binding Q9D8Y0 Nuclear protein Hcc-1 23 kd Nucleic acid binding 92 2 Q9D1J3 TCP-1-theta 59 kd Molecular chaperone 88 2 P42932 eif4e 25 kd Protein translation 61 2 P63073 Heat shock 70 kda 94 kd Molecular chaperone 86 2 P48722 protein 4L protein epsilon 29 kd Phosphoprotein binding P protein zeta/delta 28 kd Phosphoprotein binding P63101 eif-4h 27 kd Protein translation 92 1 Q9WUK2 Peroxiredoxin-1 22 kd Redox regulation P35700 HSP kd Molecular chaperone P14602 AKAP kd Sperm motility 83 2 Q protein beta/alpha 28 kd Phosphoprotein binding 73 2 Q9CQV protein sigma 28 kd Phosphoprotein binding 73 2 O70456 TG substrates Molecula Function Mascot Fragments Accession # bpq in testis r size score found Peroxiredoxin-2 21 kd Redox regulation Q61171 Glutathione S-transferase 26 kd Cell detoxification P10649 Mu 1 Peroxiredoxin-4 31 kd Redox regulation O08807 GST class-mu 5 26 kd Cell detoxification P48774 GST A kd Cell detoxification P24472 GST class-mu 6 25 kd Cell detoxificatin O35660 Poly(A)-binding protein 1 70 kd Pre-mRMA splicing P29341 Stathmin 18 kd Microtubule destabilization 72 2 P

hnrnp F 50 kd Pre-mRNA processing 128 4 Q9Z2X1 五參考文獻 Lorand, L. and Graham, R.M.

11 hnrnp F 50 kd Pre-mRNA processing Q9Z2X1 五參考文獻 Lorand, L. and Graham, R.M. (2003), Transglutaminases: crosslinking enzymes with pleiotropic functions, Nature Reviews Molecular Cell Biology 4, Nakaoka, H., et al. (1994), Gh, a GTP-binding protein with transglutaminase activity and receptor signaling function, Science 264, Fesus, L. and Piacentini, M. (2002), Transglutaminase 2: an enigmatic enzyme with diverse functions, Trends Biochem. Sci., 27, Hu, B.H. and Messersmith, P.B. (2003), Rational design of translgutaminase substrate peptides for rapid enzymatic formation of hydrogels, J. Am. Chem. Soc., 125, Ruoppolo, M., Orru, S., D smato, A., Francese, S., Rovero, P., Marino, G. and Esposito, C. (2003), Analysis of translgutaminase protein substrates by functional proteomics, Protein Sci., 12, Orru, S., Caputa. I., D Amato, A., Ruoppolo, M. and Esposito, C. (2003), Proteomics identification of acyl-acceptor and acyl-donor substrates for transglutaminase in a human intestinal epithelial cell line, J. Biol. Chem., 278, Gorman, J.J. and Folk, J.E. (1984), Structural features of glutamine substrates for transglutaminases, J. Biol. Chem., 259, Figure 1. Purification of bpq- and bpa- incorporated TG substrates from mouse testis cytosol. Mouse testis crude extract was processed by in vitro transamidation. The biotin-labeled TG substrates were purified by streptavidin beads and separated by SDS-PAGE. Polyacrylamide gel of each lane was divided equally into 20 parts and subjected to in-gel digestion and LC-MS/MS analysis. 10

Figure 2. In vitro transamidation of recombinant TG substrates. Recombinant TG substrates were processed by in vitro transamidation with biotinamido-pentylamine and biotin overlay assay.

12 Figure 2. In vitro transamidation of recombinant TG substrates. Recombinant TG substrates were processed by in vitro transamidation with biotinamido-pentylamine and biotin overlay assay. (LDH: Lactate dehydrogenase; NSFL1: NSFL1 cofactor p47; nudc: nucleo distribution protein C; translocase of the outer membrane of mitochondria; GST: Glutathione S-transferase 1; HSP70: Heat shock protein 70.2; Poly A: Poly A binding protein 1; HnrpK: Heterogeneous nuclear ribonucleoprotein K; hnrnp A2/B1: Heterogeneous ribonucleoprotein particles) 11

13 Figure 3. Western blotting of biotin-labeled TG protein substrates. 12

材料與方法

材料與方法 10-12 250-350 Sprague-Dawley rat ( SD ) 12 ( AM 8 00-PM 8 00 ) (Control group) (HS) (Heat shock group) 15 0 4 6 16 24 (PAE) ( Pulmonary air embolism group) 20 40 (25 l/min) 40 17 20 (HS&PAE) 15 4 16 20

行政院國家科學委員會補助專題研究計畫成果報告 Transglutaminase 於培養細胞對高溫及氧化壓力反應所扮演之角色 計畫類別 :X 個別型計畫 整合型計畫計畫編號 :: NSC B 執行期間 : 95 年 08 月 01 日至 96 年 07 月 31 日

行政院國家科學委員會補助專題研究計畫成果報告 Transglutaminase 於培養細胞對高溫及氧化壓力反應所扮演之角色計畫類別 :X 個別型計畫整合型計畫計畫編號 :: NSC B 執行期間 : 95 年 08 月 01 日至 96 年 07 月 31 日