臺灣博碩士論文加值系統

BRAM1(bone morphogenetic protein receptor associated molecule 1)是腫瘤抑制蛋白BS69的切割產物。它的C端和BS69C端片段有99%的相似度。BRAM1 由198 個胺基酸組成,C端具有一個MYND區域。它位於細胞質中藉由MYND 區域和Bmp接受器一號有結合的能力。由過去的實驗知道BRAM1在C.elegan的動物模式中,可能參與抑制TGFβ的訊息傳遞。但是真正的生物意義仍然未知。
為了探討BRAM1在生物中扮演的角色,我們利用斑馬魚當作動物模式,從EST資料庫分析以及5端和3端RACE的結果,選殖出斑馬魚BRAM1相關基因的cDNA。斑馬魚BRAM1同樣具有C端MYND區域,利用這段區域和斑馬魚Bmp接受器一號在生體內或生體外實驗皆證明有結合的能力。這樣結合的能力會干擾Bmp的訊息傳遞。從斑馬魚的體組織和胚胎發育的分析中顯示,斑馬魚BRAM1大量存在於母體的卵中以及胚胎初期。利用morpholinos抑制BRAM1的基因產物,導致斑馬魚胚胎在心臟和尾部的發育不正常。這樣的表現型和Bmp 變異種的表現型類似。同時注射活化Bmp接受器一號的mRNA和斑馬魚BRAM1mRNA到胚胎中,BRAM1基因產物可以抑制心臟位置從原本沒有轉置 (looping) 恢復到正常向右轉置 (D-looping)。心臟的轉置是呈現生物發育中左右不對稱基因表現的結果。所以我們利用左右不對稱基因cyclops, southpaw, lefty-1, lefty-2, 以及pitx2c。以原位雜交的方式證明這些原本分布在生物體中軸左側的基因產物, 因為BRAM1的表現降低,呈現隨意分布於中軸兩側。同時我們也分析了中軸的基因標的,發現flh和gsc的表現不正常。這些結果顯示斑馬魚BRAM1可能影響中軸的訊息傳遞進而影響左右不對稱基因的表現。
進一步在細胞株探討對BRAM1的生物機制發現,BRAM1會抑制Smad的訊息傳遞。而且從生體內實驗結果證明BRAM1和Smad1, 5, 4具有結合的能力。利用螢光染色法和共軛焦顯微鏡觀察發現BRAM1位於細胞質內,和Bmp接受器分布的位置有交集。經由Bmp ligand刺激,BRAM1會干擾Smad4和Smad5經由ligand的活化而轉移到細胞核。
從斑馬魚動物模式以及細胞株的實驗得知,BRAM1可能扮演一個負向調控Bmp生物訊息傳遞的角色。

BRAM1, bone morphogenetic protein receptor (BMPR) associated molecule 1, is a cytoplasmic protein consisting of 198 amino acids. BRAM1 shares 99% homology with the MYND domain-containing carboxy terminus of a tumor suppressor protein, BS69, which interacts with adenovirus E1A and Epstein-Barr virus-encoded nuclear protein EBNA2 through the MYND domain. Interaction of BS69 with E1A or EBNA2 inhibits viral oncoprotein-mediated transactivation. On the other hand, BRAM1 is a cytoplasmic protein and interacts with BMPR. To date, the biological role of BRAM1 remains unclear.
In this thesis work, we evaluate the physiological role of BRAM1 in zebrafish model system. zebrafish bram 1 cDNA was generated by bioinformatic search of zebrafish EST database, followed by 5’ and 3’ RACE analysis. We demonstrate that zebrafish BRAM1 associates with C-terminal region of zebrafish BMP receptor through MYND domain in vitro and in vivo. Interaction between zebrafish BRAM1 and zebrafish BMP receptor blocked BMP signaling pathway. In zebrafish, bram1 is a maternal factor and its expression profile is coordinated with bmp4 and bmp receptor in early developmental stage. bram1 was expressed throughout the embryos before blastula stage as examined by whole mount in-situ hybridization analysis, and was expressed ubiquitously in different tissues such as brain, heart, muscle, ovary, and especially ovary as examined by northern blot assay. Using antisense morpholinos to knock down BRAM1 resulted in defects in heart looping and curly tails. This phenotype of bram1 morphants was similar to phenotype of mutants involved in Bmp signaling. Injection of constitutively active bmp receptor mRNA resulted in defects in heart looping. Overexpression of BRAM1 reversed the heart position from no looping (N-looping) to rightward looping (D-looping). Further analysis of zebrafish left-right asymmetry genes incluidng cyclops, southpaw, lefty1, lefty2, and pitx2c demonstrate that BRAM1 affects the zebrafish left-right asymmetry. As zebarfish midline is critical for development of left-right asymmetry, we hypothesize that BRAM1 may have earlier effect in regulating midline. Indeed, we show that expression of flh and gsc in bram1 morphants is reduced. These findings indicate that BRAM1 may function as a negative regulator of Bmp signaling to participate in the midline development during early cell fate decision.
As Bmp signaling involves interaction of BMP and BMPR, and its downstream Smad mediators, BRAM1 may affect the BMP signaling by inhibiting Smad signaling. As tested in BALB/c3T3 and COS7 cells, mammalian BRAM1 (mBRAM1) inhibit Smad signalling pathway. mBRAM1 interacted with Bmp receptor type IA through MYND domain in the absence or presence of Bmp2 treatment. mBRAM1 interacted with Smads proteins, especially Smad4 and Smad5. Such interactions blocked the nuclear translocation of Smad4 or Smad5 in Bmp2-mediated manner. Thus, mBRAM1 inhibits Smad signaling through preventing Smad4 and Smad5 to translocate into nucleus.
In conclusion, these results suggest that BRAM1 may function as a negative regulator of BMP-mediated biological functions.

書名頁
中文摘要
英文摘要
目錄
Introduction...............................................9
I BMP signaling............................................9
I-1 BMP receptor associated protein 1 (BRAM1).............10
I-2 BMP...................................................12
I-3 BMP receptors.........................................12
I-4 BMP signaling pathways................................14
Smad signaling pathway....................................14
TAB1/TAK1/p38 MAP Kinase pathway..........................16
I-5 Negative regulations of BMP signaling pathway.........16
Inhibitory Smads (I-Smads.................................16
Receptor degradation......................................16
R-Smads degradation.......................................17
Growth factors............................................17
I-6 Significance of BMP signaling in developmental stages.17
II Zebrafish model system................................19
III Cardiac development in zebrafish.....................21
III-1 Left-right asymmetry................................22
III-2 The role of the midline barrier in L-R axis development...............................................24
Specific aims of thesis...................................26
Materials and methods.....................................27
Fish embryos..............................................27
Cell culture..............................................27
Preparation of total RNA..................................27
5'RACE and 3'RACE.........................................28
Morpholino and mRNA microinjections.......................29
Whole mount in situ hybridization.........................30
Northern blot.............................................31
Southern blot.............................................32
In vitro transcription/translation........................32
Purification of GST fusion proteins and GST pull-down assay.....................................................33
Western blot..............................................33
Knockdown assay...........................................34
Coimmunoprecipitation.....................................34
RT-PCR....................................................35
Real-time RT-PCR..........................................35
Luciferase reporter assay.................................36
Immunostaining............................................36
Yeast two-hybrid..........................................37
Results...................................................38
Part I Investigation of BRAM1 in zebrafish model
Isolation and characterization of zebrafish bram1 gene....38
Zebrafish BRAM1 interacts with zebrafish BMPR-IA in yeast.39
Zebrafish BRAM1 specifically interacts with zebrafish BMPR-IA in mammalian cells.....................................40
Zebrafish BRAM1 directly associates with zebrafish BMPR-IA........................................................41
Zebrafish BRAM1 represses the BMP signaling...............41
Temporal expression profiles of zebrafish bram1...........42
The expression pattern of zebrafish bram1.................43
Tissue distribution of zebrafish bram1....................43
Zebrafish bram1 was knocked down with antisense morpholinos in mammalian cells........................................44
Blockage of the endogenous BRAM1 results in defects of hearts and tails in zebrafish.............................44
Knockdown of BRAM1 interferes with the heart looping......45
Injections of bram1 MOs partially rescues the phenotype injecting active form BMP receptor........................46
Knockdown of BRAM1 leads to the randomization of heart jogging and heart looping.................................46
Expression of LR asymmetry genes is randomized in bram1 morphants.................................................48
BRAM1 is required for early midline development...........49
BRAM1 interferes with genes expression of midline mesoderm..................................................50
Part II Analysis of BRAM1-involved BMP signaling
mBRAM1 inhibits BMP signaling.............................50
mBRAM1 represses activity of active form BMP receptor type IA........................................................51
mBRAM1 supresses Smads signaling pathway..................51
mBRAM1 interacts with Smad4 and Smad5 and interacts with Smad1.....................................................52
Discussion................................................53
BRAM1 is a conserved protein among species................53
BRAM1 is involved in early embryonic development..........53
BRAM1 is a downstream molecule of Bmp signaling in zebrafish.................................................54
BRAM1 has a role in midline development...................55
BRAM1 functions before gastrulation stage.................56
BRAM1 inhibits BMP signaling by blocking Smads translocation.............................................56
BRAM1 may play a role in TAB1/TAK1/p38 MAP Kinase pathway.58
References................................................59
Tables....................................................71
Figures...................................................74
Appendix.................................................109
論文本文
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