脂聯素暨第一型脂聯素受體訊息傳遞之研究

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(1) . 國立臺灣師範大學生命科學系碩士論文 . 脂聯素暨第一型脂聯素受體訊息傳遞之研究 The Study of Signaling Transduction in Adiponectin/AdipoR1 Pathway. 研究生：邱柏強 Bo-Chiang Chiu. 指導教授：林炎壽博士 Yen-Shou Lin, Ph.D.. 中華民國 101 年 6 月.

(2) . 致謝時間真的過得很快，我也即將要邁向我人生的下一個階段，回想當初剛踏進實驗室的時候，對於研究生的一切都是如此的惶恐，不太確定自己想研究什麼或是能研究什麼，但是很感謝我的指導教授林炎壽老師在這段時間的傾囊相授，研究的過程當中難免遇到種種困難，也很感謝老師以及實驗室的學姐莊仁華、同學謝章亭還有學弟妹們的在技術上的協助以及精神上的互相鼓勵和扶持，因此我才能順利完成這本碩士論文。再來我要感謝我的爸爸邱茂榮、媽媽楊珠蟬以及哥哥邱信源、姐姐邱于徵，在我求學的這段期間全心全力支持我，讓我得以無後顧之憂的完成學業；還有感謝生命科學所所有教過我的老師以及別的實驗室的學長姐、同學們和一直默默地替我們服務的行政人員們，讓我能更在生命科學所這個大家庭中安穩的學習與成長。最後特別感謝師大男乙排，感謝裡面的所有成員在我求學的過程一路陪伴，我們一起聊天、一起打球、一起度過大大小小的比賽，每一座獎盃都有著許多的歡笑和淚水，我將永遠記在心頭。今天的成果，絕對是累積了很多人的幫助，要感謝的人實在太多，絕對不是三言兩語就能交代的完，只能總歸一句：謝謝大家，沒有你們就不會有今天的我。.

(3) . Contents Abstract (Chinese)............................................................................................... 1 Abstract (English) ............................................................................................... 3 Introduction ......................................................................................................... 4 Materials and methods ....................................................................................... 9 Results ................................................................................................................ 16 Discussion ........................................................................................................... 21 References .......................................................................................................... 24 Figures ................................................................................................................ 31 Appendix ............................................................................................................ 47.

(4) . 摘要脂聯素(adiponectin)是一種由脂肪組織所分泌的細胞激素 (adipokines)，由於它能促進個體對胰島素的敏感度，對於第二型糖尿病、冠狀動脈疾病以及高血壓等代謝症候群具有顯著得改善效果，因此在近幾年吸引了許多人投入研究。然而脂聯素的訊息傳遞路徑仍充滿許多未知，因此在本研究當中我們試圖找出脂聯素的訊息傳遞路徑中能與第一型脂聯素受體(AdipoR1)交互作用的傳導蛋白。我們採用了酵母雙雜交(yeast two hybrid)以及免疫沉澱(immunoprecipitation)兩種方法，其中從免疫沉澱實驗中的質譜儀定序結果中，我們找到穿膜蛋白 43(TMEM 43)。我們發現在螢光顯微鏡下可觀察到細胞中標記紅螢光蛋白的重組 TMEM43(RFP-TMEM43)與標記綠螢光蛋白的重組 AdipoR1(GFP-AdipoR1)的分布似乎有共定位的現象。進一步共免疫沉澱的實驗加以驗證也顯示了標記 FLAG 的重組 TMEM43 (FLAG-TMEM43) 與標記綠螢光蛋白的重組 AdipoR1(GFP-AdipoR1) 之間有顯著的交互作用。另外在功能方面，因為之前有報告發現 TMEM43 的表現會受到伽瑪型過氧化體增殖劑活化受器(PPAR-γ)所活化。而過氧化體增殖劑活化受器已被確定和脂聯素的訊息傳遞路徑有關，因此我們假設 TMEM43 可能透過過氧化體增殖劑活化受器的調節而涉及脂聯素的訊息傳遞路徑中。期待本研究能釐清脂聯素的訊 -1 .

(5) . 息傳遞路徑，以利於代謝症候群治療方法的未來發展。. 關鍵字：脂聯素、代謝症候群、第一型脂聯素受體、過氧化體增殖劑活化受器、穿膜蛋白 43。. -2 .

(6) . Abstract Adiponectin which is one of adipokines has attracted much attention in these years because of its significant effects to improve metabolic syndromes, such as type 2 diabetes, coronary artery disease, and hypertension. Nevertheless, the signal transduction pathway of adiponectin remains largely unknown. Utilizing two approaches, yeast two hybrid and immunoprecipitation, we began to explore the AdipoR1 binding partners which might be involved in adiponectin/AdipoR1 signaling pathway. TMEM43 was found in the immunoprecipitation recovery database of MS-SPEC results. Expression of RFP-TMEM43 and GFP-AdipoR1 in many cell types seems to colocalize under fluorescent microscope. Coimmunoprecipitation experiments were further employed to demonstrate that FLAG-TMEM43 significantly binds to GFP-AdipoR1. Previous studies considered that TMEM43 might be activated by PPAR-γ which involved in regulating cellular lipid metabolism in adiponectin/AdipoR1 signaling pathway. It is plausible that TMEM43 could play an important role in adiponectin/AdipoR1/PPARs signaling pathway. Hopefully, this study could provide new therapeutic target(s) for the drugs development to ameliorate metabolism related diseases.. Key words：adiponectin, metabolic syndromes, AdipoR1, PPARs, TMEM43.. -3 .

(7) . Introduction The prevalence of obesity and overweight has increased dramatically in recent year (Friedman, 2000, Flier, 2004). It is commonly associated with type 2 diabetes, coronary artery disease, and hypertension. The coexistence of these diseases has been termed the metabolic syndrome. To understand pathophysiological links between obesity and metabolic abnormalities becomes a critical issue. To date, adipose tissue participates in the regulation of energy homeostasis through the secretion of many biologically active adipokines, such as free fatty acid (Shulman, 2000), adipsin (White et al., 1992), leptin (Friedman, 2000), plasminogen activator inhibitor-1 (Shimomura et al., 1996), resistin (Steppan et al., 2001), and TNF-α (Hotamisligil, 1999). Therefore, it serves as an important endocrine organ in addition to being as a site of triglyceride storage and free fatty acids/glycerol release. One of such adipokines named adiponectin (also known as Acrp30, AdipoQ, ApM1, and GBP-28) has attracted much attention since 1995 (Scherer et al., 1995, Hu et al., 1996, Maeda et al., 1996, Nakano et al., 1996). Adiponectin belongs to the complement 1q family (Shapiro and Scherer, 1998). It can exist as full-length (fAd) or a smaller, globular fragment (gAd). Almost all adiponectin appear to exist as full-length adiponectin in plasma (Fruebis et al., 2001) and form wide range of multimers from low molecular weight (LMW) trimers and hexamers to high molecular weight (HMW) 12mers and 18mers (Pajvani et al., 2003, Waki et al., 2003). It is abundant in the human plasma thus accounting for approximately 0.01% of total plasma protein (Arita et al., 1999). This -4 .

(8) . concentration is three orders of magnitude higher than concentrations of most other hormones. A decrease in adiponectin level which terms hypoadiponectinemia is observed in various clinical conditions of metabolic syndrome. It was proposed that hypoadiponectinemia may be the key to the development of metabolic syndrome (Kadowaki et al., 2006). Experimental data suggest that adiponectin has important functions in the promotion of free-fatty acid oxidation and glucose uptake in mammalian cells (Berg et al., 2002, Tsao et al., 2002). Adiponectin knockout mice displayed a trend towards insulin resistance (Ziemke and Mantzoros, 2010). Administration by continuous systemic infusion of adiponectin significantly increased insulin sensitivity in type 2 diabetic mice (Yamauchi et al., 2001). In addition, adiponectin has anti-atherogenic, anti-inflammatory and cardioprotective functions observed in many pathophysiological conditions and treatment modalities. Collectively, these findings suggest that adiponectin could have an essential function in regulating whole-body energy homeostasis and be a strong candidate for the development of drugs to treat insulin resistance and type 2 diabetes.. Signal transduction of adiponectin Nevertheless, the signal transduction of adiponectin remains largely unknown. In 2003, two adiponectin receptors, AdipoR1 and AdipoR2, have been identified. These receptors are predicted to contain seven transmembrane domains, but structurally and functionally distinct from G-protein-coupled receptors (GPCR). Unlike GPCR, adiponectin -5 .

(9) . interacts with the C-termini of AdipoRs in intercellular space and the N-termini of AdipoRs are considered to interact with other molecules to begin the downstream signaling pathway in intracellcular space (Yamauchi et al., 2003a). Furthermore, APPL1 (adaptor protein containing pleckstrin homology (PH) domain, phosphotyrosine binding (PTB) domain ,and leucine zipper motif) which can mediate downstream signaling molecules, such as AMPK (Tomas et al., 2002, Yamauchi et al., 2002), PPARs (Yamauchi et al., 2003b), and MAPK (Barger et al., 2001, Michael et al., 2001, Puigserver et al., 2001), through interaction with AdipoRs in mammalian cells stimulated by adiponectin were found (Mao et al., 2006). However, no other signaling molecules that could interact with AdipoR1were further identified and defined in adiponectin/AdipoR1 signaling pathway ever since.. Transmembrane protein 43 (TMEM43) TMEM43, also named LUMA, was first identified as a integral inner nuclear membrane (INM) protein and likely interact with the nuclear lamina from a proteomics-based approach (Dreger et al., 2001). TMEM43 orthologs are widely distributed and highly conserved among all sequenced vertebrate and insect genomes, unicellular eukaryotes, some plants and even several bacteria. They also can be detected in many tissues and cell lines, such as HeLa, NIH 3T3, HEK 293T, and undifferentiated C2C12 cells (Bengtsson and Otto, 2008, Liang et al., 2011). Topologically, TMEM43 was predicted to have four -6 .

(10) . transmembrane domains (TMD1~TMD4) to span the inner nuclear membrane four times – the amino and carboxy termini of TMEM43 are both reside on the nucleoplasmic face (Bengtsson and Otto, 2008). Remarkably, they share the domain DUF 1625 in PFAM database (a database of Protein families) (Bateman et al., 2004), which has been so far only found in TMEM43 orthologs (Bengtsson and Otto, 2008). Collectively, TMEM43 is considered as an unique integral inner nuclear membrane protein to have some important and fundamental cellular functions according to its unique domains, highly conservation, and widely distribution among species and tissues. Previous studies have reported that the mutation of the encoding gene TMEM43 was related to cause some diseases, such as arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5), characterized by ventricular tachycardia, heart failure, sudden cardiac death, and fibrofatty replacement of cardiomyocytes (Merner et al., 2008), and Emery-Dreifuss muscular dystrophy (EDMD), characterized by muscular dystrophy, joint contractures, and cardiomyopathy with conduction defects (Liang et al., 2011).. Peroxisome proliferator-activated receptors (PPARs) The three PPARs (PPAR-α, PPAR-δ (also called PPAR-β), and PPAR-γ) form a subfamily of nuclear receptors. They are ligand-activated transcription factors which can directly activate gene expression through binding with peroxisome proliferator response elements (PPREs). PPARs are cellular lipid sensors which involve in the regulation of numerous -7 .

(11) . biological processes, including lipid and glucose metabolism, and overall energy homeostasis (Heald and Cawthorne, 2011). Although there are largely unknown about the function of TMEM43, previous studies have elucidated 1085 potential target genes of peroxisome proliferator-activated receptor γ (PPAR-γ) in a genome-wide identification for PPREs, including TMEM43 gene (Lemay and Hwang, 2006). TMEM43 gene containing PPRE which indicate that TMEM43 may be a part of an adipogenic pathway regulated by PPAR-γ, and perhaps dysregulation of this pathway may explain the fibrofatty replacement of the myocardium, a characteristic pathological finding in ARVC (Merner et al., 2008). PPARs are also the signaling molecules involved in adiponectin/AdipoR1 signaling pathway (Berg et al., 2001, Fruebis et al., 2001, Maeda et al., 2001, Heald and Cawthorne, 2011), and considered to regulate the expression of TMEM43. In this study we elucidated the possible signaling molecules involved in the adiponectin/AdipoR1 signaling pathway utilizing two approaches – yeast two hybrid and immunoprecipitation. We found that TMEM43 could interact with AdipoR1. Further experiments using recombinant and/or endogenous molecule verify these interactions. Possible functions of TMEM43 in the signaling pathway were also explored. Collectively, these data suggest that TMEM43 might play a role in adiponectin/AdipoR1/PPARs signaling pathway.. -8 .

(12) . Materials and Methods Yeast two hybrid － The main three steps：“yeast culture and transformation”, “yeast X-gal screening assay”, and ”yeast plasmids extraction”. I.. yeast culture and transformation：LiOAc yeast transformation. was adopted to deliver plasmids into yeast strain. Briefly, for making yeast competent cells, Y190 WT strains were incubated in 25 ml YPD broth at 30℃/240 rpm for 16~20 hrs to get optimum OD600 1.5~2.0. Six to ten fold dilution of the yeast culture (about 150 ml) which OD600 0.2~0.3 is continued to incubate at 30℃/240 rpm for 3~4 hrs to get OD600 0.4~0.6. Resuspend the yeast pellet which is collected by centrifuge at 1000 rpm for 5 mins in 20 ml “1X TE buffer + 1X LioAc (Sigma)” and incubate at 30℃/240 rpm for 1 hr. Centrifuge again at 1000 rpm for 5 mins to get yeast pellet and, resuspend in 2 ml “1X TE buffer + 1X LioAc (Sigma)” until yeast transformation. When performing the transformation, take 0.1 ml “yeast competent cell mentioned above” to mix well by pippeting with 100 μg “salmon sperm DNA (Invitrogen) which was double-boiled at 100℃ for 20 mins” and 1 μg “pAS1-CYH2 hAdipoR1 aa1~141 which will be transformed” in eppendorf, and then incubate at 30℃ for 1 hr without shaking. Add 500 μl “1X TE buffer + 1X LioAc (Sigma) + 40% PEG (Sigma)”, and incubate at 30℃ for 1 hr without shaking. Add DMSO to make it final -9 .

(13) . concentration is 10%, and mix well by pippeting. Heatshock at 42℃ for 15 mins, and then put back on ice for 1~2 mins. Centrifuge at 1000 rpm for 5 mins to get yeast pellet, and resuspend in 500 μl 1X TE buffer. Spread the suspension on YNB-Trp (BD, NJ, USA) agar plate (the kind of synthetic dropout agar plate depends on what plasmids which will be sent into yeast strain). II. yeast X-gal screening assay：While Y190 WT were transformed sequentially with two plasmids “pAS1-CYH2 hAdipoR1aa1~141” and “human skeletal muscle cDNA library contructed in pACT2” and could grow on YNB-Trp-Leu-His (BD) +3AT (SIGMA) agar plate, Transfer these colons onto a piece of filter paper to freeze and thaw by using liquid nitrogen (N2(l)). Put the piece of filter paper onto another new piece of filter paper which was impregnated beforehand with Z buffer/X-gal solution (10 ml Z buffer + 27 μl β-mercaptoethanol + 167 μl X-gal stock solution). Keep it wet and incubate at 37℃ for 20~30 mins. III. yeast plasmids extraction：Incubate the yeast clones which were positive in yeast X-gal screening assay in 5 ml YNB-Trp-Leu-His (BD) broth at 30℃/240 rpm for 16~20 hrs. Resuspend the yeast pellet which is collected by centrifuge at 25℃/13000 rpm for 1 min in 150 μl STET solution. Add 0.13 g 425~600 microns glass beads and vortex for 5 mins. Double-boil at 100℃ for 3 mins and put on ice for 5 mins. Take 100 μl supernatant to a new eppendorf after centrifuging at 4℃/13000 rpm for 10 mins, and mix well with 50 μl 7.5 M NH4OAc (SIGMA). Incubate at - 10 .

(14) . -20℃ for 1 hr. Take 100 μl supernatant to a new eppendorf after centrifuging at 4℃/13000 rpm for 10 mins, and mix well with 200 μl ice cold 100% ethanol. Incubate at -80℃ for 10 mins. Centrifuge at 4℃/13000 rpm for 10 mins, and wash the pellet twice with 500 μl 70% ethanol. Dry the pellet, and then resuspend in 10 μl sterile ddH2O.. Cell culture, transfection, and treatment － HEK 293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, N.Y., USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, South Logan, Utah, USA) and 1% penicillin-streptomycin (Sigma, St. Louis, MO, USA) in 5% CO2 atmosphere at 37 °C. For transfection, cells were replaced at a density of 3~5 × 106 per 10 cm dish and transfected 5 hrs later with a total of 10 μg of DNA and 30 μl of LipofectamineTM 2000 (Lipo2000) per dish following the manufacturer’s instruction (Invitrogen, Lohne, Germany). For adiponectin treatment, cells were seeded on 6 cm plates to get 90% confluency. Cells were serum starvation for 3 hrs before treated by adiponectin (2 ug/ml) for 10 mins. When transfection using calcium phosphate precipitation approach, HEK 293T cells were seeded at a density of 3~5 × 106 per 10 cm dish for 16~24 hrs incubation. Aliquot 0.5 ml 2X HBS into a sterile eppendorf. Ten μg DNA, 60 μl 2 M CaCl2 and enough distilled water to bring the total volume to 0.5 ml in another eppendorf. Add the CaCl2/DNA mix to the HBS slowly with pipetting, and mix gently by bubbling during the addition. Add directly to cells by dropping slowly and evenly into - 11 .

(15) . medium, trying to cover as much of the plate as possible. Incubate in 5% CO2 atmosphere at 37 °C for 16~24 hrs. Refresh the medium and resume incubation for 24 ~48 hrs.. Adiponectin preparation － The resource of adiponectin is purified from bacterium expression. The BL21 transformed with pGEX FLAG-FLAG-Adiponectin were incubated in LB broth supplemented with 100 μg/ml ampicillin at 37℃/240 rpm for 16~18 hrs to get OD600 0.9~1.0. One hundred fold dilution of the bacterial culture which OD600 about 0.1 is continued to incubate at 37℃/240 rpm for 2~3 hrs. Final concentration 500 μm of IPTG (Isopropyl β-D-1-thiogalactopyranoside) is added to induce the expression of pGEX FLAG-FLAG-Adiponectin in BL21 until the bacterial culture get OD600 0.4~0.5. The bacteria broth is continued to incubate at 37℃/240 rpm for 5 hrs. Bacteria pellet is collected by centrifuge and kept in -80℃ until use. To purify FLAG-FLAG-adiponectin, bacteria were lysed in bacteria lysis buffer (10 mM HEPES, 50 mM NaCl, 50 mM β-glycerolphosphate). The mixture was votexed, and sonicated, followed by centrifuged at 4℃/13000 rpm for 30 mins. The supernatant were incubated with anti-FLAG-conjugated agrose beads (Sigma) at 4℃ for 16 hrs, washed by bacteria lysis buffer, and eluted the FLAG-FLAG-adiponectin by FLAG peptide dissolved in bacteria lysis buffer. The purified FLAG-FLAG-adiponectin was kept in -80℃ until use. - 12 .

(16) . Identification of binding partners of AdipoR1 － HEK 293T cells were transfected with pCMV5 FLAG-AdipoR1. Cells were lysed by lysis buffer (20 mM Tris-base pH7.9, 20 mM NaCl, 1 mM EDTA, 5 mM EGTA, 20 mM β-glycerolphosphate, 1 mM DTT, 1 mM PMSF, 1 tablet protease inhibitor/50 ml, 20 nM calyculin A, 0.25% CHAPS) to pull down FLAG-AdipoR1 in vivo through immunoprecipitation and resolved by 10% SDS-PAGE, and silver stained. The differential regions between control and experimental groups were cutting down and the protein sequences were identified by MS-SPEC. MS-SPEC was carried out by Academia Sinica.. Immunoprecipitation and Western blot analysis － Cells were washed once with PBS and lysed by different lysis buffers：buffer A (20 mM Tris-base pH7.9, 20 mM NaCl, 1 mM EDTA, 5 mM EGTA, 20 mM β-glycerolphosphate, 1 mM DTT, 1 mM PMSF, 1 tablet/50 ml protease inhibitor, 20 nM calyculin A, 0.25% CHAPS) or buffer B which is the same components as buffer A except use 0.5% triton X-100 instead of 0.25% CHAPS. The total protein volumes were quantified by using Bio-Rad protein assay (BIO-RAD, Hercules, CA, USA) to detect OD600. For immunoprecipitation, total lysates were incubated with anti-FLAG-conjugated agrose beads (Sigma) or magnetic GFP-Trap-M (ChromoTek, Martinsried, Germany) at 4℃ for 2 hrs, washed by lysis buffer containing 0.5 M LiCl, and analyzed by Western blot. - 13 .

(17) . For Western blot analysis, total lysates were prepared in sample buffer, heated at 95℃ for 10 mins, resolved by 10% SDS-PAGE, and immunoblotted with primary and HRP-conjugated secondary antibodies. Immunoblots within the linear range were scanned on Fuji imager. Monoclonal anti-FLAG (Sigma), monoclonal anti-GFP (Santa cruz, Santa Cruz, CA, USA) were used as primary antibody. HRP-conjugated anti-rabbit (Jackson, West Grove, PA, USA) and anti-mouse (Jackson) were used as secondary antibody.. Lentivirus-mediated RNA interference (RNAi)－ TMEM43 shRNAs (A1： 5’-CCTCAACCTTATGACACGGATCTCGAGATCCGTGTCATAAG GTTGAGG-3’ and B1： 5’-GAGGTGTTTCATAGAGAACTACTCGAGTAG TTCTCTATGAAACACCTC-3’), control luciferase shRNA(5’-CGCTGAGT ACTTCGAAATGTCCTCGAGGACATTTCGAAGTACTCAGCG-3’) in the pLKO.1-puro vector for knockdown, and the helper vector (pCMV5-△8.91 and pCMV5-pMD.G) were purchased from National RNAi Core Facility. HEK 293T cells were cotransfected with pLKO.1-puro vector and the helper vector via calcium phosphate. After 24, 48, and 72 hrs of incubation, medium containing lentivirus were harvested and centrifuged at 4℃/25000 rpm for 2 hrs to get the lentivirus - 14 .

(18) . pellet, and the pellet was resuspended in 1X PBS (the volume of 1X PBS depends on the size of lentivirus pellet). For lentivirus transduction, HEK 293T cells were seeded at a density of 3~5 × 105 per 6 cm dish for 16~24 hrs incubation and the lentivirus were added with 4 μg/ml polybrene. The transduced cells were then selected by puromycin (3 μg/ml) to get stable cell clones.. Transactivation assay (luciferase assay) － In expression of PPARs experiment, cells were transfected by pα(H-H)-pGL3, containing the -1664 to +83 region of the human PPARα gene promoter (a generous gift from Dr. Bart Staels, Institut Pasteur de Lille), pGL3-γ1p3000, pGL3-γ2p1000, pGL3-γ3p800 (generous gifts from Dr. Johan Auwerx, École polytechnique fédérale de Lausanne), pGL3-δp735, and pGL3-basic respectively with phRG-TK for 24~48 hrs. After experimental treatment, cells were treated following the manufacturer’s instruction (Promega, WI, USA). Wash once with PBS and lysed by 1X PLB. Predispense 20 μl of LARII into luminometer tube. Transfer 2 μl of PLB lysate, and mix well by vortex. Measure firefly luciferase activity by luminometer immediately. Dispense 20 μl of Stop & Glo® Reagent, and mix well by vortex. Measure Renilla luciferase activity.. - 15 .

(19) . Results YTH35aa interact with the amino terminus of AdipoR1 in yeast two hybrid system. －. AdipoR1 was predicted to be an integral plasma. membrane protein and have seven transmembrane domains belongs to G-protein-coupled receptors family (GPCR)；however, it is structurally and functionally distinct from GPCR (Yamauchi et al., 2003a). The carboxy terminus is intercellular to interact with adiponectin and the amino terminus is intracellular to bind with other downstream signaling molecule, such as APPL1(Mao et al., 2006). Therefore, the amino terminus of AdipoR1 aa1~141 was constructed as a bait protein to screen human skeletal muscle cDNA library. We found 28 yeast clones could grow on YNB-Trp-Leu-His plate, however 18 out of 28 these yeast clones were positive in X-gal screening assay as shown in Fig. 1. After bioinformatic analysis, one the most strongest signal clones was found to be only translated into 35 amino acids. We named it YTH35aa protein/peptide. Due to the concerns that YTH35aa is not an endogenous protein and expresses as an extremely small peptide, we temporally postponed this direction. Its application as an activator or inhibitor due to its strong binding will be considered further in the near future.. AdipoR1 not only distributes over plasma membrane, but also at intracellular space －. AdipoR1 was generally considered to localize. at plasma membrane as it’s the receptor of adiponectin. It was also mentioned that recombinant AdipoR1 can be detected at the intracellular - 16 .

(20) . organelles via immunocytochemistry (Yamauchi et al., 2003a). We used fluorescence microscopy to observe the distribution of recombinant GFP tagged AdipoR1 in HEK 293T cells, C2C12 myoblasts (Fig. 2A and 2B), and RD myoblasts(data not shown). It shows a distribution pattern outside of nuclei. We also examined the localization of endogenous AdipoR1 in RD myoblasts by using immunocytochemistry. As sown in Fig. 2C, which is similar to the pattern in Fig. 2A and 2B, AdipoR1 was found that not only distributes over plasma membrane described as previous studies, but also in intracellular space. It’s localization might indicate some other important roles in addition to adiponection/AdipoR1 signaling pathway.. A possible candidate previously named TMEM43 had a high score in MS-SPEC sequencing results. －. In previous studies, AdipoR1 can. be detected in kidney through Northern blot analysis (Yamauchi et al., 2003a). Therefore HEK 293T, a human embryonic kidney, was a cellular model chosen to study adiponectin/AdipoR1 signaling pathway. AMPK in HEK 293T cells indeed can be activated when treated with adiponectin for 5~15 mins (Fig. 3A). It also indicates that the recombinant adiponectin purified from bacteria is functional. Thereafter, we use HEK 293T as well as C2C12 myoblast as cellular models to continue our investigation. In order to search for possible partners in adiponection/AdipoR1 signaling pathway, immunoprecipitation were adopted to identify candidate proteins. FLAG-AdipoR1 was overexpressed in HEK 293T - 17 .

(21) . cells and the lysate was subjected to FLAG antibody immunoprecipitation. After running onto SDS-PAGE, one of differentiated bands about 44 kDa was found and the gel was sent for MS-SPEC analysis(Fig. 3B). One of the candidate proteins, named transmembrane protein 43 (TMEM43), was identified. The cDNA encoding TMEM43 was then cloned from human skeletal muscle cDNA library into pCMV5 and pDsRed-Monomer-C1 eukaryotic expressing vectors.. AdipoR1 interacts with TMEM43. －. First of all, the association. between AdipoR1 and TMEM43 was verified. HEK 293T cells were cotransfected with RFP-TMEM43 and/or GFP-AdipoR1. As shown in Fig. 4A, RFP-TMEM43 seems to locate at the peripheral of nuclei. When cells were cotransfected with RFP-TMEM43 and GFP-AdipoR1, they both colocalized around the nuclear rim (Fig. 4B). In immunocytochemistry, we also found that endogenous AdipoR1 could localize with RFP-TMEM43 at similar position in RD myoblasts as shown in Fig. 4C. In order to further confirm the association between AdipoR1 and TMEM43, HEK 293T cells were cotransfected with FLAG-TMEM43 and GFP-AdipoR1. FLAG-TMEM43 was immunoprecipitated by anti-FLAG-conjugated agrose beads. As shown in Fig. 5, GFP-AdipoR1 can only be co-immunoprecipitated by FLAG-TMEM43, but not by control group. In the same experiment, FLAG-MST1, a cytosolic protein, was used as a negative control. Meanwhile, FLAG-mTOR 1362~2549 cotransfected with GFP-LST8 was - 18 .

(22) . included as a positive control in the same experiment (Long et al., 2005). This association was further proved by reciprocal immunoprecipitation. Thus, the lysates were immunoprecipitated by magnetic GFP-Trap-M, run onto SDS-PAGE, and blotted by FLAG antibody in Western blot analysis. GFP-empty was introduced and the other arrangement of control groups are similar to Fig. 5. As shown in Fig. 6, majority of FLAG-TMEM43 was co-immunoprecipitated by GFP-AdipoR1 although a trace of FLAG-TMEM43 still associated with GFP-empty. Furthermore, endogenous TMEM43 could be remarkably detected in the FLAG-AdipoR1 immunoprecipitation, but not in the immunoprecipitation of FLAG-empty or FLAG-LST8 (Fig. 7).. Overexpression or suppression of TMEM43 would not change the adiponectin/AdipoR1 signaling pathway in HEK 293T cells. －. Due. to the lack of functional reports in the literatures and wonder it’s possible role in adiponectin/AdipoR1 signaling pathway, we initially surveyed several signaling molecules, such as AMPK, MAPK, Akt, 4E-BP, and caspase-3 via Western blot, either by gain of function or loss of function approaches. As shown in Fig. 8A, commercial antibody well recognizes endogenous and recombinant TMEM43. Moreover, RNAi of TMEM43 effectively knockdown its protein expression level (Fig. 8B). However, there was no significant change no matter TMEM43 was overexpressed (Fig. 8C) or suppressed (Fig. 8D) in HEK 293T cells. Although there are largely unknown about the function of TMEM43, TMEM43 is considered as a part of an adipogenic pathway owing to the - 19 .

(23) . transcription of TMEM43 gene containing PPRE is regulated by PPAR-γ (Lemay and Hwang, 2006). The remarkable interaction between AdipoR1 and TMEM43 in our previous data indicates that TMEM43 could play a crucial role in the AdipoR1/PPARs pathway. Initially, we considered TMEM43 as an upstream signaling molecule of PPARs. Neither overexpression of recombinant TMEM43 nor suppression of endogenous TMEM43 would change the expression of PPARs-luciferase assay without adiponectin treatment (data not shown). Luciferase expression induced by FLAG-p53 via binding to p53RE was introduced as a control to confirm transactivation assay was usable (Fig. 8E). On contrast, we examined the possibility of TMEM43 as a downstream signaling molecule of PPARs. The transcription of TMEM43 gene could be regulated by PPARs as mentioned in previous studies (Lemay and Hwang, 2006). However, there was no significant change of abundance in expression of TMEM43 via Western blot analysis (data not shown).. - 20 .

(24) . Discussion The signal transduction pathway of adiponectin/AdipoR1 is considered to be a key position in improving metabolic syndromes, such as type 2 diabetes, coronary artery disease, and hypertension. However, it still remains largely unknown so far. We began to clarify this pathway and explore the AdipoR1 binding partners which might mediate in adiponectin/AdipoR1 signaling pathway. Currently, TMEM43 and YTH35aa were found in approaches of immunoprecipitation and yeast two hybrid, respectively. Fluorescent images have shown TMEM43 that a colocalized pattern with AdipoR1 in HEK 293T cells and RD myoblasts. In the further verification, the binding between TMEM43 and AdipoR1 was significant and specific through our coimmunoprecipitation experiments, even using other lysis buffers containing different detergents such as triton X-100 and MEGA-10 (data not shown). Since TMEM43 mainly locates at nuclei rim, this finding extends an original hypothesis that binding partners of AdipoR1 might only be closed to plasma membrane or within the cytoplasmic. As mentioned before, AdipoR1 not only distributes over plasma membrane, but also at intracellular space. The interaction between TMEM43 and AdipoR1 might indicate some other important roles in addition to adiponection/AdipoR1 signaling pathway. In yeast two hybrid approach, YTH35aa significantly interacted with the amino terminus of AdipoR1 aa1~141 through selective medium and X-gal screening assay in vitro. The sequence encoding YTH35aa was subcloned from pACT2 to pCMV5 HA and pEGFP-C1 for further - 21 .

(25) . binding assay and functional examination in eukaryotic system in vivo. However, YTH35aa is not a endogenous protein and is too small to be detected in Western blot, even using Tricin-SDS-PAGE instead of Glycin-SDS-PAGE. On the other hand, GFP tag might be too large for YTH35aa peptide and the original distribution and function of YTH35aa in vivo might be influenced. Based on these concern；therefore, we temporally postponed this direction. Nevertheless, due to YTH35aa strongly bound to the amino terminus of AdipoR1 aa1~141, we hypothesized it might be functional to be an activator or inhibitor that can regulate the signaling transduction in adiponectin/AdipoR1 signaling pathway. We will further validate this possibility in the near future. How can TMEM43 protein related to the pathway of adiponectin/AdipoR1 signaling？ Previous studies have shown that TMEM43 is highly conserved and widely distributed among species and tissues, and predicted to be an unique integral inner nuclear membrane protein. Although the mutation of the encoding gene TMEM43 was related to cause some diseases, such as ARVC5 (Merner et al., 2008), and EDMD (Liang et al., 2011), the functions of TMEM43 still remain largely unknown so far. We initially surveyed several signaling molecules, such as AMPK, MAPK, Akt, 4E-BP, and caspase-3, but there was no significant change no matter TMEM43 was overexpressed (Fig. 8C) or suppressed (Fig. 8D) in HEK 293T cells. On the other hand, TMEM43 was considered as a part of an adipogenic pathway owing to the transcription of TMEM43 gene containing PPRE which is regulated by PPAR-γ (Lemay and Hwang, 2006). PPARs were showed to be involved - 22 .

(26) . in Adiponectin/AdipoR1 signaling pathway (Yamauchi et al., 2003b). Therefore, the physiological roles of TMEM43 involved in adiponectin/AdipoR1/PPARs signaling pathway were further investigated. Currently, we focus on changes of the activity of PPARs and free-fatty acid oxidation in HEK 293T cells while TMEM43 was overexpressed or suppressed and with or without adiponectin stimulation. Topologically, TMEM43 was predicted to have four transmembrane domains (TMD1~TMD4) to span the inner nuclear membrane four times. The amino and carboxy termini of TMEM43 are both reside on the nucleoplasmic face (Bengtsson and Otto, 2008) and contain the PFAM domain DUF1625 which has been so far only found in TMEM43 orthologs (Bateman et al., 2004). The region (domain) of TMEM43 responsible for interacting with AdipoR1 will be mapped. Previous studies demonstrated that APPL1 interacts with AdipoR1 via its PTB domain (phosphotyrosine binding domain). Therefore, we will survey other endogenous proteins which might contain similar sequence of PTB domain to interact with AdipoR1 from proteomic database. The outcomes demonstrated that TMEM43 associated with AdipoR1. The critical role of TMEM43 in the adiponectin/AdipoR1/PPARs signaling pathway for adipogenic metabolism will be further elucidated. Besides, YTH35aa might have application on the regulation of the signaling transduction in adiponectin/AdipoR1 pathway to be an activator or inhibitor. The novel discovery might provide a new therapeutic approaches for the development of drugs so as to treat metabolism related diseases. - 23 .

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(33) . adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med 7:941-946. Ziemke F, Mantzoros CS (2010) Adiponectin in insulin resistance: lessons from translational research. Am J Clin Nutr 91:258S-261S.. - 30 .

(34) . Figures. Fig. 1. - 31 .

(35) . Fig. 1. YTH35aa interacts with the amino terminus of AdipoR1. aa1~141 in yeast two hybrid system － AdipoR1 aa1~141 was constructed into pAS1-CYH2. Human skeletal muscle cDNA library were used to screen the possible binding proteins. Twenty-eight clones were scored and 18 out of 28 clones were positive in X-gal screening assay. The first clone (indicated by blue box) which is the most strongest signal clone can be translated into 35 amino acids and was named YTH35aa.. - 32 .

(36) . (A). (B). (C). Fig. 2. - 33 .

(37) . Fig. 2. AdipoR1 distributes over plasma membrane as well as. intracellular space － (A) HEK 293T cells and (B) C2C12 myoblasts were transfected with plasmids which express GFP tagged on either N-terminus or C-terminus of AdipoR1. Fluorescence images were recorded by using confocal fluorescence microscope SP2-DMRE. The same field was observed after nuclei staining. (C)The localization of endogenous AdipoR1 in RD myoblasts using immunocytochemistry. After fixation and blocking, cells were incubated with anti-AdipoR1 polyclonal antibody followed by fluorescein isothiocyanate (FITC)-conjugated secondary antibodies. Cells were then incubated with DAPI for 15 mins and imaged umder the fluorescence microscope.. - 34 .

(38) . (A). (B). Fig. 3. - 35 .

(39) . Fig. 3. TMEM43 was discovered from immunoprecipitation. experiments accompanied with MS-SPEC methods － (A) Phospho-AMPK T172 can be activated by adiponectin in HEK 293T cells. HEK 293T cells were stimulated with adiponectin (Ad) (2 μg/ml) for 5~15 mins after 3 hrs serum starvation. 2DG (25 mM) was used as control for examining activated AMPK. β-actin was detected as a loading control. The intensity of phospho-AMPK T172 signal was quantified by free software NIH IMAGE J. The value of lanes 5~8 were normalized to lane 5 and indicated at the bottom of top panel. (B) FLAG-AdipoR1 WT was overexpressed in HEK 293T cells. The immunoprecipitation of FLAG antibody fllowed by intensively wash was resolved by 10% SDS-PAGE, and silver stained. The gels were sent for MS-SPEC analysis and one of possible candidates, TMEM43, was obtained (indicated by red box). The bottom panel is the result of immunoprecipitation analyzed by Western blot analysis to detect the expression of FLAG-RFP and FLAG-AdipoR1 WT. The immunoprecipitation is representative of one experiment that was repeated 4~5 times.. - 36 .

(40) . (A). (B). (C). Fig. 4. - 37 .

(41) . Fig. 4. TMEM43 was colocalized with AdipoR1 around nuclear rim. － (A) HEK 293T cells were simply transfected with plasmid expressed RFP-TMEM43. Red fluorescent signals were observed around nuclear rim under fluorescence microscope. (B) HEK 293T cells were cotransfected with RFP-TMEM43 and GFP-AdipoR1 using Lipo2000. Cells were also stained with DAPI. Images were merged and arranged at last column. (C) RD myoblasts transfected with RFP-TMEM43 were fixed followed by permeabiling. Endogenous AdipoR1 detected using immunocytochemistry approach and RFP tagged TMEM43 signal were both imaged under fluorescence microscope.. - 38 .

(42) . Fig. 5. - 39 .

(43) . Fig. 5. FLAG-TMEM43 interacts with GFP-AdipoR1 － HEK 293T. cells were cotransfected by FLAG-TMEM43 and GFP-AdipoR1 via Lipo2000 and lysed in lysis buffer containing 0.25% CHAPS. Lysates were treated as indicated in “Materials and Methods”. FLAG-TMEM43 was immunoprecipitated by anti-FLAG-conjugated agrose beads followed by SDS-PAGE and Western blot analysis. FLAG-MST1 was used as a negative control. Meanwhile, FLAG-mTOR 1362~2549 cotransfected with GFP-LST8 was included as a positive control. Each blot is representative of one experiment that was repeated 3~4 times.. - 40 .

(44) . Fig. 6. - 41 .

(45) . Fig. 6. Reciprocal immunoprecipitation demonstrated. GFP-AdipoR1 interacts with FLAG-TMEM43 － HEK 293T cells were cotransfected by FLAG-TMEM43 and GFP-AdipoR1 via Lipo2000 and lysed in lysis buffer containing 0.25% CHAPS. Lysates were treated as indicated in “Materials and Methods”. GFP-AdipoR1 was immunoprecipitated by magnetic GFP-Trap-M beads followed by SDS-PAGE and Western blot analysis. FLAG-MST1 is used as a negative control. Meanwhile, FLAG-mTOR 1362~2549 cotransfected with GFP-LST8 was included as a positive control.. - 42 .

(46) . Fig. 7. - 43 .

(47) . Fig. 7. Endogenous TMEM43 interacts with FLAG-AdipoR1 －. HEK 293T cells were simply transfected by FLAG-AdipoR1. FLAG-AdipoR1 was immunoprecipitated by anti-FLAG-conjugated agrose beads followed by SDS-PAGE and Western blot analysis. FLAG-LST8 was used as a negative control. Each blot is representative of one experiment that was repeated 3~4 times.. - 44 .

(48) . (A). (B). (C). (D). (E). Fig. 8 - 45 .

(49) . Fig. 8. TMEM43 knowdown or overexpression do not affect AMPK. or Akt signaling－ (A) FLAG-TMEM43 was transfected into HEK 293T cells and antibodies were used to examine both endogenous and recombinant proteins expression. (B) HEK 293T cells were infected with lentivirus particles containing TMEM43 RNAi to knockdown endogenous TMEM43. RNAi of luciferase was used as a control. (C) HEK 293T cells were transfected with plasmids which were indicated on the figure. (D) RNAi approach of TMEM43 were adopted. In both (C) and (D), signaling molecules such as pT172 AMPK, AMPK, pT202/204 MAPK, MAPK, pS473 Akt, Akt, cleaved caspase-3, procaspase-3, etc were examined. Each blot is representative of one experiment that was repeated 4~5 times. (E) HEK 293T cells were cotransfected with plasmids which were indicated on the figure. The cells were lysed for luciferase assay followed manufacture’s instruction. Protein expression was showed on the left panel. The fold of stimulation in FLAG-p53 compared to FLAG-empty on the p53 promoter was calculated on the right panel.. - 46 .

(50) . Appendix － solutions. 10X TE buffer：0.1 M tris-HCl, 10 mM EDTA, adjust to pH7.5, autoclave.. 10X LiOAc：1 M LiOAc, adjust to pH7.5 with acetic acid, autoclave.. 50% PEG：50% polyethylene glycol 3350, autovlave.. Z buffer：0.06 M Na2HPO4‧7H2O, 0.04 M NaH2PO4‧H2O, 0.01 M KCl, 0.001 M MgSO4‧7H2O, adjust to pH7.0, autoclave.. X-gal stock solution：20 μg/μl 5-bromo-4-chloro-3-indolyl-β-Dgalactopyranoside (X-gal) in N,N-dimethyformamide (DMF).. STET solution：8% sucrose, 50 mM tris-HCl pH8.0, 50 mM EDTA, 5% triton X-100.. 2X HBS：40 mM Hepes pH7.05, 274 mM NaCl, 10 mM KCl, 1.4 mM Na2HPO4, 12 mM Dextrose, filter through a 0.22 μm filter.. - 47 .

(51) . Appendix － antibodies Antibody. Brand. Catalog. Conc. (Titer). FLAG. SIGMA. －. 1：1000. HA. －. －. 1：1000. GFP. EPITOMICS. #1533-S. 1：1000. AdipoR1. PHOENIX. G-001-45. －. AMPKα-1. EPITOMICS. #1596-S. 1：1000. pAMPKα (Thr172). Cell Signaling. #2535. 1：1000. MAPK. Cell Signaling. #9212. 1：1000. pMAPK (pT202/204). EPITOMICS. #2219-1. 1：1000. S6K. Santa Cruz. Sc-230. 0.2 μg/μl. pS6K (Thr389). Cell signaling. #9205. 1：1000. AKT. EPITOMICS. #1085-S. 1：1000. pAKT (pS473). EPITOMICS. #2118-S. 1：1000. Pro-Caspase-3. SANTA CRUZ. Sc-7148. 0.2 μg/μl. Cleaved-Caspase-3. Cell Signaling. #9661. 1：1000. β-actin. SIGMA. A5441. 1：2000. - 48 .

(52)