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沙库巴曲缬沙坦治疗急性心肌梗死合并Ⅱ～Ⅲ期慢性肾脏病患者的短期效果

·临床研究·基金项目：安徽省安庆市科技局基金项目（２０２１Ｚ２００１）作者简介：沈国秀，主治医师，Ｅｍａｉｌ：１１５８７１０５２９＠ｑｑ．ｃｏｍ通信作者：项学军，主任医师，Ｅｍａｉｌ：ｇｕａｎｇｆ４５０８＠１６３．ｃｏｍ

沙库巴曲缬沙坦治疗急性心肌梗死合并Ⅱ～Ⅲ期慢性肾脏病患者的短期效果

沈国秀，王贤进，顾崇怀，项学军安庆市立医院心血管内科，安庆２４６０００

［摘要］　目的　探讨沙库巴曲缬沙坦治疗急性心肌梗死合并Ⅱ～Ⅲ期慢性肾脏病患者住院期间的治疗效果。方法　回顾性分析２０１９年１月１日至２０２２年３月３１日于安庆市立医院住院的急性心肌梗死合并Ⅱ～Ⅲ期慢性肾脏病患者病历资料，依据其使用不同种类肾素－血管紧张素－醛固酮系统（ＲＡＡＳ）抑制剂将其分组为沙库巴曲缬沙坦组９６例，缬沙坦组１２６例，共计２２２例患者。分别记录入院及出院时估算的肾小球滤过率（ｅＧＦＲ）、左室射血分数（ＬＶＥＦ），疗效值及安全效应值分别为出院ＬＶＥＦ、ｅＧＦＲ水平与入院时比较的差值，同时使用１∶１倾向性评分匹配（ＰＳＭ）比较均衡基线资料后效应量的改变。结果　２组出院ＬＶＥＦ、ｅＧＦＲ较入院时均有改善，差异有统计学意义（Ｐ＜０．０５），组间比较显示沙库巴曲缬沙坦组出院ＬＶＥＦ、ｅＧＦＲ改善程度更大，差异有统计学意义（Ｐ＜０．０５）；

在进行ＰＳＭ均衡基线后（匹配后２组各７０例），沙库巴曲缬沙坦组出院ＬＶＥＦ、ｅＧＦＲ较入院时仍有改善，差异有统计学意义（Ｐ＜０．０５）；缬沙坦组出院ＬＶＥＦ较入院时有改善，差异有统计学意义（Ｐ＜０．０５），ｅＧＦＲ水平出院与入院比较，差异无统计学意义（Ｐ＞０．０５）；ＰＳＭ后ＬＶＥＦ改善程度２组比较，差异无统计学意义（Ｐ＞０．０５）。结论　沙库巴曲缬沙坦能够改善急性心肌梗死合并Ⅱ～Ⅲ期慢性肾脏病患者住院期间心、肾功能，使用安全有效。［关键词］　心肌梗死；沙库巴曲缬沙坦；肾功能不全ＤＯＩ：１０．３９６９／Ｊ．ｉｓｓｎ．１６７２６７９０．２０２２．０３．０１７

G蛋白耦联胆汁酸受体激动剂INT777通过激活AMPK信号通路抑制施万细胞成髓鞘过程

南通大学学报(医学版)Journal of Nantong University (Medical Sciences) 2021 : 41 (1)6・D0I:10.16424/32-1807/r.2021.01.002G 蛋白耦联胆汁酸受体激动剂INT777通过激活AMPK 信号通路抑制施万细胞成髓鞘过程*关晋东：丁杰，刘晓宇，孙诚**** ［基金项目］国家自然科学基金青年基金资助项目(81701222)** ［作者简介］关晋东，男，汉族，生于1995年10月，山西省晋城市人，硕士在读，研究方向:外周神经发育及损伤修复机制的研究。

*** ［通信作者］孙诚，电话**************,E-mail: ********************.cn(南通大学教育部/江苏省神经再生重点实验室/神经再生协同创新中心，南通226001)［摘要］目的：研究G 蛋白耦联胆汁酸受体(G-protein-coupled bile acid receptor 1, GPBAR1,同时也被称为TGR5) 特异性激动剂6琢-乙基-23(S)-甲基胆酸［6 alpha-ethyl-23(S)-methylcholic acid, INT777［对原代施万细胞髓鞘形成的影响并初步探讨其可能的作用机制遥方法:用5 滋mol/L 的INT777处理二丁酰环腺苷酸(dibutyryl cyclic adenoslne phosphate, dbcAMP )诱导分化原代施万细胞成髓鞘模型，用免疫印迹(Western Blot )方法检测髓磷脂蛋白表达量的变化遥同时，提取施万细胞总核糖核酸后用定量聚合酶链式反应试验检测INT777对髓鞘形成过程相关分子基因表达的影响遥另外，用Western Blot 方法检测INT777对单磷酸腺苷活化蛋白激酶/核糖体蛋白S6激酶(adenosine 5'-monophosphate-activatedprotein kinase/ribosomal S6 kinase, AMPK/S6K )信号途径的影响遥结果:在dbcAMP 诱导分化原代施万细胞成髓鞘过程中，5 滋mol/L INT777的处理抑制了髓鞘早期生长因子20,八聚体结合转录因子6,髓磷脂蛋白的表达，且5滋mol/L INT777 处理激活了 AMPK 的活性，抑制了雷帕霉素作用靶点信号通路遥结论：INT777抑制dbcAMP 诱导的施万细胞成髓鞘过程，这种抑制作用可能是通过激活施万细胞AMPK 活性、抑制S6K 活性实现的遥［关键词］施万细胞曰髓鞘曰6琢-乙基-23(S)-甲基胆酸曰单磷酸腺苷活化蛋白激酶［中图分类号］R338.1 ［文献标志码］A ［文章编号］1674-7887(2021)01-0006-05G-protein-coupled bile acid receptor agonists INT777 inhibits myelination of Schwann cells byactivating AMPK signaling pathway*GUAN Jindong **, DING Jie, LIU Xiaoyu, SUN Cheng ***('Key Laboratory of Neuroregeneration of Jiangsu and Ministry ofEducation, Co-innovation Center of Neuroregeneration, Nantong University, Nantong 226001)［Abstract ］ Objective: To investigate the effects of G-protein-coupled bile acid receptor 1(GPBAR1, also known as TGR5)specific agonist 6 alpha-ethyl-23(S)-methylcholic acid(INT777) on myelination in primary Schwann cells and the underlying mechanisms. Methods: Primary Schwann cells were treated with 5 滋mol/L INT777 and dibutiryl cyclic adenoslne phosphate (dbcAMP), and the changes of myelin protein zero were detected by Western Blot. Meanwhile, the total RNA was extractedfrom Schwann cells and quantitative real time polymerase chain reaction was employed for detecting myelin gene expression. In addition, the effect of INT777 on the adenosine 5' -monophosphate -activated protein kinase/ribosomal protein S6 kinase (AMPK/S6K) signaling pathway was examined by Western Blot. Results : Treatment with 5 滋mol/L INT777 inhibited theexpression of myelin early growth response -2, octamer -binding transcription factor 6, and myelin protein zero during dbcAMP-induced myelination of differentiated Schwann cells, and treatment with 5 滋mol/L INT777 activated AMPK activity and inhibited mTOR signaling pathway. Conclusion: INT777 attenuates dbcAMP-induced myelination of Schwann cells, whichmay be achieved by activating AMPK and inhibiting S6K activity.［Key words ］ Schwann cell; myelination; 6 alpha-ethyl -23(S)-methylcholic acid; adenosine 5'-monophosphate-activated protein kinase外周神经系统(peripheral nervous system, PNS) 在机体内分布广泛且起到介导靶器官与中枢神经系统信号传递的重要作用。

血管内皮间质样转分化遗传示踪小鼠模型的构建及其在肝纤维化研究中的应用

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Western blot 验证不同足细胞系标志蛋白的相对表达
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mTOR通路

Seminars in Cell &Developmental Biology 36(2014)79–90Contents lists available at ScienceDirectSeminars in Cell &DevelopmentalBiologyj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /s e m c dbGrowing knowledge of the mTOR signaling networkKezhen Huang a ,Diane C.Fingar a ,b ,∗aDepartment of Cell and Developmental Biology,University of Michigan Medical School,Ann Arbor,MI 48109-2200,United StatesbDivision of Metabolism,Endocrinology,and Diabetes (MEND),Department of Internal Medicine,University of Michigan Medical School,Ann Arbor,MI 48109-2200,United Statesa r t i c l ei n f oArticle history:Available online 19September 2014Keywords:mTOR mTORC1mTORC2InsulinAmino acids Energya b s t r a c tThe kinase mTOR (mechanistic target of rapamycin)integrates diverse environmental signals and trans-lates these cues into appropriate cellular responses.mTOR forms the catalytic core of at least two functionally distinct signaling complexes,mTOR complex 1(mTORC1)and mTOR complex 2(mTORC2).mTORC1promotes anabolic cellular metabolism in response to growth factors,nutrients,and energy and functions as a master controller of cell growth.While signiﬁcantly less well understood than mTORC1,mTORC2responds to growth factors and controls cell metabolism,cell survival,and the organization of the actin cytoskeleton.mTOR plays critical roles in cellular processes related to tumorigenesis,metabolism,immune function,and aging.Consequently,aberrant mTOR signaling contributes to myriad disease states,and physicians employ mTORC1inhibitors (rapamycin and analogs)for several pathological conditions.The clinical utility of mTOR inhibition underscores the important role of mTOR in organismal physiology.Here we review our growing knowledge of cellular mTOR regulation by diverse upstream signals (e.g.growth factors;amino acids;energy)and how mTORC1integrates these signals to effect appropriate downstream signaling,with a greater emphasis on mTORC1over mTORC2.We highlight dynamic sub-cellular localization of mTORC1and associated factors as an important mechanism for control of mTORC1activity and function.We will cover major cellular functions controlled by mTORC1broadly.While signif-icant advances have been made in the last decade regarding the regulation and function of mTOR within complex cell signaling networks,many important ﬁndings remain to be discovered.©2014Elsevier Ltd.All rights reserved.Contents 1.Introduction ..........................................................................................................................................802.Growth factor sensing by the TSC–Rheb axis ........................................................................................................812.1.Insulin-PI3K-Akt signaling ....................................................................................................................812.2.EGF-Ras-MAPK signaling......................................................................................................................822.3.mTORC2regulation ...........................................................................................................................822.4.Negative feedback signaling ..................................................................................................................823.Amino acid sensing by the Rag–Ragulator axis and other emerging factors ........................................................................823.1.The Rag GTPases ..............................................................................................................................833.2.The Ragulator complex .......................................................................................................................833.3.The v-ATPase ..................................................................................................................................833.4.Emerging amino acid sensing factors .........................................................................................................844.The lysosome as a critical platform for upstream signal integration and mTORC1activation ......................................................855.Energy and stress sensing ............................................................................................................................856.Major cellular functions controlled by mTORC1.....................................................................................................86∗Corresponding author at:Department of Cell and Developmental Biology,University of Michigan Medical School,Ann Arbor,MI 48109-2200,United States.Tel.:+17347637541.E-mail address:dﬁngar@ (D.C.Fingar)./10.1016/j.semcdb.2014.09.0111084-9521/©2014Elsevier Ltd.All rights reserved.80K.Huang,D.C.Fingar/Seminars in Cell&Developmental Biology36(2014)79–907.Future directions (87)Acknowledgments (87)References (87)1.IntroductionAll cells from single-celled organisms to those comprising mul-ticellular organisms sense and respond rapidly toﬂuctuations in their nutritional and energetic environments in order to modulate cell metabolism appropriately and maintain cellular homeostasis. Consequently,cells coordinate nutritional and energetic supply and demand tightly to prevent engagement in ATP-consuming anabolic processes when environmental resources become limited. During evolution,multicellular organisms acquired the additional ability to sense and respond to long-range systemic signals(i.e. hormones;growth factors;mitogens;cytokines)(referred to col-lectively as“growth factors”here)to enable communication between tissues and organ systems.mTOR,the mechanistic tar-get of rapamycin,functions as a critical integrator of these diverse environmental cues by integrating them into appropriate cellu-lar responses.mTOR,an evolutionarily conserved serine/threonine protein kinase,belongs to the phosphatidylinositol-3kinase(PI3K)-related kinase(PIKK)superfamily.mTOR represents the functional target of a natural macrolide antibiotic called rapamycin(clini-cally known as sirolimus).Rapamycin,produced by the bacterium Streptomyces hygroscopicus,was discovered in soil samples from Easter Island(known as Rapa Nui to the native population)in the 1970s[1,2].Rapamycin reduces eukaryotic cell proliferation to var-ious degrees,with immune cells showing strong sensitivity.To identify the target of rapamycin,Hall and colleagues performed an elegant genetic screen in1991in the budding yeast Saccha-romyces cerevisiae.Mutations in three genes,Fpr1(an orthologue of FKBP12[FK506-binding protein12]),Tor1and Tor2,conferred resis-tance to rapamycin[3](and reviewed in[2]).Today we understand that rapamycin associates with an endogenous cellular protein, FKBP12,and this complex docks to the FRB(FKBP12rapamycin binding)domain located immediately N-terminal to the C-terminal mTOR kinase domain(see the accompanying article for greater detail regarding mTOR structure),resulting in allosteric inhibi-tion of mTOR activity and signaling[4–6].By afﬁnity puriﬁcation of FKPB12-rapamycin binding proteins,several groups identiﬁed the mammalian orthologue of budding yeast Tor1/2in1994–1995 [7–9].mTOR constitutes the catalytic core of two known signal-ing complexes,mTOR complex1(mTORC1)and mTOR complex2 (mTORC2)[10,11].These mTOR complexes(mTORCs)possess dis-tinct substrates,cellular functions,and sensitivity to rapamycin. Acute rapamycin treatment inhibits cellular mTORC1but not mTORC2signaling while longer-term rapamycin treatment sup-presses mTORC2function by compromising complex integrity to variable degrees depending on cell type[12].Rapamycin fails to inhibit the phosphorylation of all mTORC1substrates equally [13,14].It completely inhibits phosphorylation of S6K1(ribosomal protein S6kinase1)but only partially inhibits phosphorylation of 4EBP1(eukaryotic initiation factor4E binding protein1).The devel-opment of ATP-competitive mTOR catalytic inhibitors(i.e.Torin1; Ku-0063794)revealed that mTORC1phosphorylates substrates in both rapamycin-sensitive and-insensitive manners[15–18],pos-sibly due to differential substrate access to the kinase active site controlled by the mTOR FRB domain[19]and/or due to differen-tial substrate quality conferred by phosphorylation site consensus sequence[20,21].The exclusive mTOR partner raptor(regulatory-associated pro-tein of mTOR)deﬁnes mTORC1[22,23]while the exclusive mTOR partner rictor(rapamycin-insensitive companion of mTOR)deﬁnes mTORC2[24,25](Fig.1).In addition to raptor,mTORC1contains mLST8/G␤L(mammalian lethal with Sec13protein8/G-protein ␤-protein subunit like)[26],PRAS40(Akt/PKB substrate40kDa) [27,28],and deptor(DEP-domain-containing mTOR interacting protein)[29].Raptor serves a scaffolding role,functioning to recruit substrates to the mTOR kinase through their TOS(TOR signal-ing)motifs[30,31].Global deletion of raptor in mice results in early embryonic lethality(e5.5)[32],similar to the global knock-out of mTOR[33].PRAS40and deptor function as both suppressors and targets of mTORC1,likely by acting as competitive sub-strates[29,34],while mLST8/G␤L is not essential for mTORC1 function[32].In addition to rictor[24,25],mTORC2contains mSIN1 (mammalian stress-activated protein kinase interacting protein1) [35,36],protor1/2(protein observed with rictor1/2)(aka PRR5) [37],mLST8/G␤L[24,25],and deptor[29].Thus,mTORC1and mTORC2contain distinct and shared partner proteins.Similar to raptor within mTORC1,rictor and mSin1serve as critical scaffolds that control mTORC2integrity,regulation by upstream signals, and substrate choice[24,25,35,36].Unlike mTORC1,mLST8/G␤L is required for mTORC2function;like rictor,its deletion in mice causes embryonic lethality(e10.5)[32].The role of protor remains unclear.It is important to note that mTOR also assembles into relatively homologous TORC1and TORC2complexes in bud-ding andﬁssion yeast,underscoring the ancestral origin of the TORCs.While TORC1in yeast responds simply to environmental nutri-ents and energy,mTORC1in higher eukaryotes responds to a broader array of upstream signals,integrating cues from growth factors(i.e.insulin;IGF;EGF;cytokines)to modulate cellular func-tions appropriately(Fig.1)[10,11].mTORC1function absolutely requires sufﬁcient levels of amino acids such that their withdrawal inactivates mTORC1signaling rapidly and renders mTORC1acti-vation refractory to virtually all other inputs,including growth factors.To limit cellular engagement in energy costly anabolic pro-cesses,nutrient and growth factor withdrawal as well as diverse types of cell stress(i.e.low energy;hypoxia;ER stress;ROS(reac-tive oxygen species))downregulate mTORC1signaling[38].Upon activation,mTORC1signaling drives cap-dependent protein syn-thesis,cell growth,and cell proliferation through phosphorylation of the ribosomal protein S6kinases(S6K1/2)and the eukaryotic ini-tiation factor4E(eIF4E)binding proteins1–3(4EBP1-3)at least in part[10,11,39].While the current set of direct mTORC1substrates remains somewhat limited(e.g.S6Ks;4EBPs;IRS-1;ULK1;Lipin1; TFEB;Grb10),quantitative phosphoproteomic screens identiﬁed a large number of downstream mTORC1effectors,many of which likely represent bonaﬁde mTORC1substrates[40,41](Fig.1). mTORC1promotes other anabolic processes including lipid and nucleotide synthesis and suppresses autophagy,a degradative pro-cess in which autophagosomes break down macromolecules and organelles during nutrient and energy starvation.Thus,mTORC1 drives anabolic and suppresses catabolic cellular processes.Our understanding of the regulation and function of mTORC2lags far behind that of mTORC1due to its more recent discovery[24,25] and the lack of mTORC2-speciﬁc inhibitors.Growth factors activate mTORC2,which phosphorylates a limited set of known substrates including Akt(aka PKB),PKC␣(protein kinase C␣),and SGK1(serum and glucocorticoid-induced protein kinase).mTORC2modulates cell metabolism and the organization of the actin cytoskeleton and enhances cell survival,due to its activation of the survival kinase Akt[42,43](Fig.1).K.Huang,D.C.Fingar /Seminars in Cell &Developmental Biology 36(2014)79–9081Fig.1.Regulation of the mTORC1and mTORC2signaling network by diverse upstream inputs.Growth factors such as insulin or EGF activate mTORC1through either the PI3K-Akt or the Ras-MAPK (ERK)-RSK axes,respectively.Growth factor-mediated activation of mTORC1absolutely requires sufﬁcient levels of amino acids,which are sensed through a variety of factors,as indicated.mTORC1action also requires sufﬁcient levels of energy (i.e.ATP)and/or oxygen,which are sensed by AMPK,REDD1,and TCA cycle metabolites (i.e.␣KG).The TSC complex integrates diverse upstream signals to regulate mTORC1action.TSC suppresses the conversion of Rheb-GDP to Rheb-GTP,a small GTPase that activates mTORC1.mTORC1phosphorylates a limited known set of bona ﬁde substrates to drive anabolic and suppress catabolic cellular processes and to mediate negative feedback toward PI3K.Growth factors (i.e.insulin)also activate mTORC2through poorly deﬁned signaling intermediates.Aberrant mTORC1function contributes to myriad patho-logic conditions including cancer and benign tumor syndromes,metabolic disorders (e.g.type II diabetes;obesity),cardiovascu-lar disorders,inﬂammatory disorders,and neurological disorders [10,11].Consequently,clinicians employ rapamycin (aka sirolimus)and rapamycin analogs (rapalogs)(i.e.everolimus;temsirolimus)for immunosuppression following renal transplantation and for treatment of renal cell carcinoma,neuroendocrine tumors of pan-creatic origin,tuberous sclerosis complex (TSC,a benign tumor syndrome),and cardiac restenosis following angioplasty [6].The role of mTOR in pathophysiology of disease combined with the utility of mTORC1inhibitors in clinical medicine underscores the importance of elucidating the regulation and function of mTORC1at the cellular level [44].2.Growth factor sensing by the TSC–Rheb axisGrowth factors,in particular insulin/IGF (insulin-like growth factor)and EGF (epidermal growth factor),represent the best understood inputs that lead to activation of mTORC1upon converg-ing on the TSC (tuberous sclerosis complex)/Rheb axis [45].TSC,composed of TSC1(aka hamartin),TSC2(aka tuberin),and a morerecently discovered third subunit,TBC1D7(TBC [Tre2-Bub2-Cdc16]1domain family member 7),functions as a tumor suppressor that inhibits mTORC1[46](Fig.1).Inactivating mutations in either TSC1or TSC2increases mTORC1signaling and causes an autosomal dominant disease in which benign tumors form in various organs including brain,kidney,and heart [47].TSC inhibits mTORC1by inhibiting Rheb (Ras homolog enriched in brain),a small Ras-like GTPase essential for mTORC1activation by all upstream signals.2.1.Insulin-PI3K-Akt signalingInsulin/IGF binding to its cognate cell surface tyrosine kinase receptor leads to tyrosine phosphorylation of IRS (insulin receptor substrate)proteins,which recruits and activates PI3K (phos-phatidylinositol 3-kinase)[48](Fig.1).Increased production of the phospho-lipid PI(3,4,5)P3on lipid membranes by PI3K recruits Akt via its PH (pleckstrin homology)domain,leading to PDK1-mediated phosphorylation of Akt on its activation loop (T308)[49].It is important to note that additional phosphorylation of Akt on its hydrophobic motif (S473)by PI3K-controlled mTORC2boosts Akt activity several fold further [49,50].Activated Akt then phosphorylates TSC2on several sites (S939;T1462)to82K.Huang,D.C.Fingar/Seminars in Cell&Developmental Biology36(2014)79–90suppress the inhibitory effect of the TSC complex toward mTORC1, thus leading to increased mTORC1signaling[51,52].TSC2pos-sesses a GAP(GTPase activating protein)domain that hydrolyzes active Rheb-GTP to inactive Rheb-GDP.Thus,in response to insulin/PI3K signaling,Akt phosphorylates and inactivates TSC2, which increases Rheb-GTP loading and mTORC1kinase activity [27].While Rheb-GTP interacts with the mTOR kinase domain [27,53],the underlying molecular mechanism by which Rheb acti-vates mTORC1remains unclear.Other parallel mechanisms contribute to activation of mTORC1 by insulin/IGF.Akt and mTORC1phosphorylate PRAS40,an mTORC1inhibitory partner(on T246and S183/S212/S221,respec-tively),inducing the dissociation of PRAS40from mTORC1and thus relieving PRAS40-mediated substrate competition[27,28,34,54]. Insulin/PI3K signaling also leads to mTORC1-mediated phospho-rylation of raptor(on S863)to promote mTORC1signaling[55,56]. Moreover,phosphorylation of mTOR itself(on S1261,S2159,and T2164)by unknown kinases[57,58]and on S1415by IKK␣[59] contributes to increased mTORC1signaling.While mTOR autophos-phorylation on S2481plays no known role in mTORC1function, it serves as a biomarker for mTORC1and mTORC2catalytic activ-ity in intact cells[5].Many phospho-proteomic studies agree that mTOR and its partner proteins undergo phosphorylation on many sites[40,41,60].Consequently,a challenge for the future will be to identify the kinases that act on these sites directly and to deci-pher the regulation and functional signiﬁcance of complex mTORC phosphorylation.2.2.EGF-Ras-MAPK signalingEGF activates mTORC1signaling independently of the PI3K/Akt axis.EGF binding to its cell surface tyrosine kinase receptor acti-vates the Ras GTPase,which leads to activation of c-Raf,MEK (MAPK/ERK kinase),MAPK(mitogen activated protein kinase) (aka ERK)and RSK(p90ribosomal S6kinase(Fig.1).Similar to Akt,MAPK and RSK phosphorylate TSC2on different sites (S540/S644and S1798,respectively)to suppress the inhibitory action of TSC2toward Rheb[61,62].By a parallel pathway,the Ras/MAPK pathway converges on raptor.MAPK phosphorylates raptor(on S8/S696/S863)and RSK phosphorylates raptor(on S719/S721/S722)[63,64]to promote mTORC1signaling.2.3.mTORC2regulationInsulin/PI3K signaling leads to mTORC2-mediated phosphory-lation of Akt on its hydrophobic motif(HM)site,S473(Fig.1)as well as the HM sites of other AGC kinases,PKC␣(on S657),and SGK1(on S422)[25,50,65].Insulin increases the kinase activity of mTORC2in vitro in a manner sensitive to cellular treatment with the PI3K inhibitor wortmannin[66].Thus,insulin/PI3K signaling activates mTORC2;it is important to note,however,that the sig-naling intermediates that link PI3K to mTORC2remain virtually unknown.Interestingly,while TSC inhibits mTORC1,TSC activates mTORC2[66].MEFs lacking TSC2display reduced mTORC2kinase activity toward Akt in vitro and decreased Akt S473phosphoryla-tion in intact cells in a manner independent of the well-known mTORC1-mediated negative feedback loop that attenuates PI3K signaling(discussed below)[66].In addition,TSC associates with mTORC2.These data suggest quite different regulation of mTORC2 compared to mTORC1.On the other hand,the Rac1GTPase interacts with mTOR and provides an activating signal to both mTORC1and mTORC2in response to growth factors in a PI3K/Akt independent manner,suggesting that common upstream inputs co-regulate both mTORCs[67].There is no question that important discoveries await regarding mTORC2regulation.As mTORC2mediates Akt S473phosphorylation,it would seem that mTORC2lies upstream of mTORC1.While such epistasis may hold true in certain cellular contexts,genetic knockout or knockdown of core mTORC2components(i.e.rictor;mSin1)in many cultured cell types has no effect on TSC2phosphoryla-tion and mTORC1signaling[32,68].Thus,mTORC2function is not required for mTORC1action.On the other hand,mTORC2function is required for Akt-mediated phosphorylation of other substrates (i.e.FoxO1/3a)[32,68].These data can be explained by the known essential requirement for activation loop site(T308)but not HM site(S473)phosphorylation for Akt kinase activity;Akt S473phos-phorylation boosts Akt activity further and may modulate substrate speciﬁcity[32,49,68].Thus,in many cellular contexts Akt phospho-rylation on its activation loop without HM-site phosphorylation provides sufﬁcient activity to mediate downstream signaling to mTORC1.Interestingly,mTORC2associates with ribosomes in a growth factor sensitive manner[69,70].Structurally intact ribosomes,but not protein synthesis itself,are required for mTORC2kinase activity in vitro and signaling in intact cells[70].Thus,a direct interaction of mTORC2with ribosomes may play a role in insulin/PI3K-meditated mTORC2activation.mTORC2also promotes turn-motif (TM)site phosphorylation of Akt(T450)and several conventional PKCs(PKC␣T638and PKC␤T641)in a co-translational manner independently of growth factor status,functioning to increase protein stability and folding[71,72].In addition to interacting with ribosomes(likely those associated with ER engaged in pro-tein translation),mTORC2associates with an ER sub-compartment called MAM(mitochondrial-associated ER membrane)in a growth factor stimulated manner.mTORC2inactivation decreases MAM integrity,mitochondrial metabolism,and cell survival[73].2.4.Negative feedback signalingSeveral negative feedback mechanisms modulate the mTOR signaling network,as signal attenuation limits signal amplitude and duration critical for homeostatic control of complex biolog-ical systems.Cellular TSC loss leads to elevated and constitutive mTORC1signaling independent of growth factor status and atten-uates PI3K signaling,thus producing a state of cellular insulin resistance[74,75].S6K1and mTORC1phosphorylate IRS-1directly to induce its degradation,thus uncoupling the insulin receptor from PI3K.mTORC2also limits PI3K signaling by inducing IRS-1 degradation[76].mTORC2phosphorylates and stabilizes Fbw7,an ubiquitin ligase subunit that targets IRS-1for degradation[76]. Grb10was identiﬁed as a direct mTORC1substrate in phospho-proteomic screens[40,76].mTORC1-mediated phosphorylation of Grb10,a growth factor receptor-bound adaptor that limits growth factor signaling,stabilizes Grb10and attenuates both PI3K and MAPK/ERK signaling.Depending on cellular context,either S6K1 or Akt phosphorylate mSin1directly(on T86and T398),a crit-ical mTORC2partner,dissociating mSin1from the complex and decreasing mTORC2signaling[40].Along similar lines,several groups reported that S6K1phosphorylates rictor directly(T1135) [77–80],which may reduce mTORC2signaling to Akt[78,79].These data reveal that both mTORC1and mTORC2engage in negative feedback to maintain proper signaling by growth factor receptors and the mTORCs.3.Amino acid sensing by the Rag–Ragulator axis and other emerging factorsSufﬁcient levels of amino acids,particularly leucine,are essen-tial for basal mTORC1signaling from yeast to mammals and for robust activation of mTORC1in response to growth factor signalsK.Huang,D.C.Fingar/Seminars in Cell&Developmental Biology36(2014)79–9083Fig. 2.Rag heterodimers recruit mTORC1to lysosomal membranes for Rheb-mediated activation.Activation of mTORC1by amino acids through Rag GTPase heterodimers involves the v-ATPase,Ragulator complex,and Rag regulatory factors. The Ragulator complex,which acts as a GEF toward RagA/B GTPases,induces forma-tion of active RagA/B GTP–RagC/D GDP heterodimers.The GATOR1complex functions as a GAP(GTPase activating protein)for RagA/B(thus inhibiting Rag heterodimers) while folliculin(FLCN)and its associated proteins(FNIP1/2)functions as a GAP for Rag C/D(thus promoting a Rag heterodimer active state).The GATOR2complex sup-presses GATOR1.Active RagA/B GTP–RagC/D GDP heterodimers bind mTORC1through raptor to recruit mTORC1to the lysosomal surface where Rheb resides.When loaded with GTP,Rheb activates mTORC1through a poorly deﬁned mechanism.An“inside-out”model proposes that the v-ATPase and Ragulator respond to amino acid levels inside the lysosomal lumen to control Rag nucleotide binding state.in higher eukaryotes[81,82].How cells sense amino acid levels remains poorly deﬁned,but great progress has been made in recent years identifying the machinery that propagates amino acid sens-ing proximal to mTORC1.While several signaling molecules that link amino acid sensing to mTORC1have been identiﬁed(see text below),the Rag GTPases,the Ragulator complex,and the v-ATPase represent the best-characterized links between amino acid sensing and mTORC1.3.1.The Rag GTPasesThe evolutionarily conserved family of Rag GTPases function as obligate heterodimers in which Rag A or Rag B dimerizes with RagC or RagD(Fig.2).Upon amino acid stimulation,RagA/B loads with GTP and binds raptor while RagC/D loads with GDP[83,84]. Expression of dominant-active RagA/B mutants(loaded consti-tutively with GTP)promote mTORC1signaling in the absence of cellular amino acids while expression of dominant-negative RagA/B mutants(nucleotide-free)suppress mTORC1signaling in the presence of amino acids.Thus,heterodimers composed of RagA/B GTP–RagC/D GDP form during amino acid sufﬁciency to promote mTORC1signaling and heterodimers composed of RagA/B GDP–RagC/D GTP form during amino acid withdrawal[83,84]. As exogenous expression of RagA/B GTP–RagC/D GDP heterodimers more strongly activate mTORC1in amino acid deprived cells than exogenous expression of dominant-active RagA/B alone,these data suggest that the nucleotide-bound state of RagC/D as well as that of RagA/B indeed contributes to mTORC1signaling in response to amino acids.How do Rags control amino acid-mediated activation of mTORC1?While Rheb-GTP provided in vitro increases mTORC1 kinase activity directly[27],active RagA/B GTP–RagC/D GDP het-erodimers provided in vitro are insufﬁcient[83].Cellular amino acid stimulation induces the translocation of mTOR and raptor from a poorly deﬁned cytoplasmic compartment to LAMP1/2-positive lysosomal membranes,a site to which the Rags and Rheb also localize[83,85].Importantly,mTORC1translocation requires the Rags[83].These data suggest a model in which the amino acid-Rag axis activates mTORC1by controlling mTORC1subcel-lular localization:amino acid signaling drives the formation of active RagA/B GTP–RagC/D GDP heterodimers,which bind raptor to recruit mTORC1to the lysosomal surface where mTORC1receives an essential activating input from Rheb(and possibly from other inputs)(Fig.3).While Rheb docks to internal membranes by a farnesyl lipid moiety,Rag GTPases do not possess lipid-anchoring motifs.3.2.The Ragulator complexA pentameric complex called the Ragulator,consisting of p18 (LAMTOR1),p14(LAMTOR2),MP1(LAMTOR3),c7orf59(LAMTOR4), and HBXIP(hepatitisB virus X interacting protein)(LAMTOR5),was found to anchor the Rags to lysosomal membranes through p18 myristoylation[85,86](Fig.3).The Ragulator complex not only tethers the Rags to lysosomal membranes,it possesses GEF activity toward RagA/B to enable exchange of GDP for GTP,thus converting Rag heterodimers to an active state[85].Conversely,a complex of proteins termed GATOR1,which contains proteins DEPDC5,Nprl2, and Nprl3,binds Rag heterodimers and possesses GAP activity for RagA/B,thus converting Rag heterodimers to an inactive state[87].A complex called GATOR2,which contains proteins Mios,Wdr24, Wdr59,Seh1L,and Sec13,suppresses GATOR1.Indeed,inactivation of GATOR1subunits renders mTORC1resistant to amino acid depri-vation while inactivation of GATOR2subunits suppresses mTORC1 signaling[87].RagC/D not only function as obligate binding partners for RagA/B,their nucleotide binding state also participates in amino acid controlled mTORC1function.Folliculin(FLCN)and its inter-acting partners FNIP1/2are required for amino acid-stimulated translocation of mTORC1to lysosomes,where they dock to Rag GTPases in the absence of amino acids,poised to convert RagC/D GTP to RagC/D GDP upon docking of mTORC1to the lysosomal sur-face upon amino acid addition[88,89](Fig.2).FLCN possess GAP activity toward RagC/D but not RagA/B and thus converts Rag heterodimers from an inactive RagA/B GDP–RagC/D GTP to an active RagA/B GTP–RagC/D GDP nucleotide-bound state that stabilizes mTORC1docking[89].The discovery that a spatial-temporal mech-anism governs amino acid-mediated mTORC1signaling explained a long-standing mystery in theﬁeld regarding why growth factors fail to activate mTORC1in the absence of amino acids:if mTORC1local-izes within the cell to the wrong place at the wrong time,mTORC1 cannot be activated by upstream inputs.3.3.The v-ATPaseHow do cells sense amino acid levels?The discovery that the v-ATPase(vacuolar H+-adenosine triphosphatase)interacts with the Ragulator complex and Rag GTPases on the lysosomal surface and senses amino acids levels,possibly from within the lysoso-mal lumen,begins to elucidate these important but poorly resolved questions[90].v-ATPase subunits and its ATP hydrolyzing catalytic activity are required for generation of active RagA/B GTP–RagC/D GDP heterodimers,for amino acid-mediated localization of mTORC1to lysosomes,and for activation of mTORC1(Fig.2).Its classical func-tion as a proton pump that lowers luminal pH of lysosomes is not required for mTORC1activation,however[90].Amino acids modulate interactions between the v-ATPase,Ragulator,and Rags [90],and amino acid accumulation within the lysosomal lumen correlates with recruitment of mTORC1to lysosomal membranes. Thus,mTORC1appears to sense intra-lysosomal amino acids by an“inside-out”mechanism.It is important to note,however, that the overall importance of intra-lysosomal amino acids for mTORC1recruitment remains unclear.In this proposed model,。

以FXR为核心的胆汁酸代谢机制研究进展

Hans Journal of Biomedicine 生物医学, 2018, 8(4), 62-68Published Online October 2018 in Hans. /journal/hjbmhttps:///10.12677/hjbm.2018.84008Advanced Progression in the Mechanism of Bile Acid Metabolism Targeting FXRXiuli Yang1, Sicong Tian1, Bo Pang1, Baolong Li2, Yujuan Shan1*1Department of Food Science and Engineering, Harbin Institute of Technology, Harbin Heilongjiang2Center of Drug Safety Evaluation, Heilongjiang University of Chinese Medicine, Harbin HeilongjiangReceived: Oct. 4th, 2018; accepted: Oct. 19th, 2018; published: Oct. 26th, 2018AbstractBile acids are important physiological factors that facilitate the digestion & absorption of dietary lipids and fat-soluble vitamins in the gut. In addition, they also act as signaling molecules to regu-late glucose homeostasis, lipid metabolism and energy expenditure. Disorders of bile acid meta-bolism can lead to a series of diseases. The nuclear receptor farnesoid X receptor (FXR) is a spe-cific bile acid receptor which plays an important role in the metabolism of bile acids through the regulation of multiple metabolic pathways and of corresponding target genes. Consequently, FXR is targeted to be a new drug for the therapy of disorders related to bile acid metabolism. This ar-ticle reviews the recent progressions of FXR in regulating bile acid metabolism and its mechanism, which aims to provide scientific strategies for the prevention/treatment of bile acid metabolic disorders, and new drugs exploration.KeywordsBile Acids, Farnesoid X Receptor, Bile Acid Metabolism以FXR为核心的胆汁酸代谢机制研究进展杨修利1，田思聪1，庞博1，李宝龙2，单毓娟1*1哈尔滨工业大学食品科学与工程系，黑龙江哈尔滨2黑龙江中医药大学药物安全性评价中心，黑龙江哈尔滨收稿日期：2018年10月4日；录用日期：2018年10月19日；发布日期：2018年10月26日*通讯作者。

抑制还是转导：信号分子调节机体健康与疾病

生命科学Chinese Bulletin of Life Sciences第22卷第3期

2010年3月Vol. 22, No. 3

Mar., 2010

文章编号：1004-0374(2010)03-0240-08抑制还是转导：信号分子调节机体健康与疾病裴　钢1,2

(1 同济大学，上海 200092；2 中科院上海生命科学研究院生物化学与细胞生物学研究所，上海200031)

摘　要：细胞内的信号转导网络是由多条功能特异且彼此关联的信号通路所构成，它们赋予了细胞功能的多样性和可塑性，同时也必须受到精细严谨的调控。一些功能广泛的信号调节因子，如β-抑制蛋白(β-arrestin)，在细胞信号转导网络完整性的维持中扮演着重要的角色。β-arrestin分子的经典功能是终止G-蛋白偶联受体(G-protein-coupled receptors)下游信号转导，即受体脱敏，但最近许多研究证据表明，这种脱敏功能(负调控)还可以针对其他的信号转导途径。例如，β-arrestin能够通过不同的机制负调控三条重要的NF-κB激活通路，该功能异常则导致NF-κB持续激活以及下游炎性因子的过度分泌。此外，近年来发现β-arrestin还能作为支架蛋白介导功能性信号复合物的形成。例如，在特定外界信号刺激下，β-arrestin 1能够转移至细胞核内并与组蛋白乙酰化酶p300相互作用而调控基因表达。该机制的生理意义之一反映在多发性硬化症的小鼠模型中，β-arrestin1在发病小鼠中较正常小鼠表达上调并能够显著加重病情。与之相反，在细胞质中富集的β-arrestin2参与了胰岛素激活时InsR/Akt/β-arrestin2/Src信号复合体的形成，它的缺失能够导致胰岛素耐受和2型糖尿病的发生。因此，在特定的条件下，β-arrestin对于胞内信号的传递究竟是抑制还是激活，已成为细胞信号转导中的关键问题，并在机体健康和疾病状态的相互转化中的起着重要作用。关键词：β-arrestin；受体脱敏；信号转导；NF-κB信号通路；核定位；胰岛素信号复合体中图分类号：R329.2；R363.2

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ＲＥＶＩＥＷ　ｗｗｗ．ｎａｔｕ　ｒｅ．ｃｏｍ／ａｊａ　

Ｒｏｌｅ　ａｎｄ　ｒｅｇｕｌａｔｉｏｎ　ｏｆ　ＥＧＦＲ　ｉｎ　ａｃｔｉｎ　ｒｅｍｏｄｅｌｉｎｇ　ｉｎ　ｓｐｅｒｍ　ｃａｐａｃｉｔａｔｉｏｎ　ａｎｄ　ｔｈｅ　ａｃｒｏｓｏｍｅ　ｒｅａｃｔｉｏｎ　

Ｈａｈｎ　Ｂｒｅｉｔｂａｒｔ　ａｎｄ　Ｎｉｒ　Ｅｔｋｏｖｉｔｚ　Ｔｏ　ｂｉｎｄ　ａｎｄ　ｆｅｒｔｉｌｉｚｅ　ｔｈｅ　ｅ器，ｔｈｅ　ｓｐｅｒｍａｔｏｚｏｏｎ　ｓｈｏｕｌｄ　ｕｎｄｅｒｇｏ　ｆｅｗ　ｂｉｏｃｈｅｍｉｃａＩ　ａｎｄ　ｍｏｔｉｌｉｔｙ　ｃｈａｎｇｅｓ　ｉｎ　ｔｈｅ　ｆｅｍａｌｅ　ｒｅｐｒｏｄｕｃｔｉｖｅ　ｔｒａｃｔ　ｃｏｌｌｅｃｔｉｖｅｌｙ　ｃａｌｌｅｄ　ｃａｐａｃｉｔａｔｉｏｎ．Ｔｈｅ　ｃａｐａｃｉｔａｔｅｄ　ｓｐｅｒｍａｔｏｚｏｏｎ　ｂｉｎｄｓ　ｔｏ　ｔｈｅ　ｅｇｇ　ｚｏｎａ　ｐｅｌｌｕｃｉｄａ，ａｎｄ　ｔｈｅｎ　ｕｎｄｅｒｇｏｅｓ　ｔｈｅ　ａｃｒｏｓｏｍｅ　ｒｅａｃｔｉｏｎ　（ＡＲ），ｗｈｉｃｈ　ａｌｌｏｗｓ　ｉｔｓ　ｐｅｎｅｔｒａｔｉｏｎ　ｉｎｔｏ　ｔｈｅ　ｅ器．Ｔｈｅ　ｍｅｃｈａｎｉｓｍｓ　ｒｅｇｕｌａｔｉｎｇ　ｓｐｅｒｍ　ｃａｐａｃｉｔａｔｉｏｎ　ａｎｄ　ｔｈｅ　ＡＲ　ａｒｅ　ｎｏｔ　ｃｏｍｐｌｅｔｅｌｙ　ｕｎｄｅｒｓｔｏｏｄ．　Ｉｎ　ｔｈｅ　ｐｒｅｓｅｎｔ　ｒｅｖｉｅｗ。ｗｅ　ｓｕｍｍａｒｉｚｅ　ｓｏｍｅ　ｄａｔａ　ｒｅｇａｒｄｉｎｇ　ｔｈｅ　ｒｏｌｅ　ａｎｄ　ｒｅｇｕｌａｔｉｏｎ　ｏｆ　ｔｈｅ　ｅｐｉｄｅｒｍａｌ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ　ｒｅｃｅｐｔｏｒ（ＥＧＦＲ）ｉｎ　ｔｈｅｓｅ　ｐｒｏｃｅｓｓｅｓ．Ｉｎ　ｔｈｅ　ｃａｐａｃｉｔａｔｉｏｎ　ｐｒｏｃｅｓｓ，ｔｈｅ　ＥＧＦＲ　ｉｓ　ｐａｒｔｉａｌｌｙ　ａｃｔｉｖａｔｅｄ　ｂｙ　ｐｒｏｔｅｉｎ　ｋｉｎａｓｅ　Ａ（ＰＫＡ）．ｒｅｓｕｌｔｉｎｇ　ｉｎ　ｐｈｏｓｐｈｏｌｉｐａｓｅ　Ｄ（ＰＬＤ）　ａｃｔｉｖａｔｉｏｎ　ａｎｄ　ａｃｔｉｎ　ｐｏｌｙｍｅｒｉｚａｔｉｏｎ．Ｐｒｏｔｅｉｎ　ｋｉｎａｓｅ　Ｃ　ａｌｐｈａ（ＰＫＣａ），ｗｈｉｃｈ　ｉｓ　ａｌｒｅａｄｙ　ａｃｔｉｖａｔｅｄ　ａｔ　ｔｈｅ　ｂｅｇｉｎｎｉｎｇ　ｏｆ　ｔｈｅ　ｃａｐａｃｉｔａｔｉｏｎ。ａｌｓｏ　ｐａｒｔｉｃｉｐａｔｅｓ　ｉｎ　ＰＬＤ　ａｃｔｉｖａｔｉｏｎ．Ｆｕｒｔｈｅｒ　ａｃｔｉｖａｔｉｏｎ　ｏｆ　ｔｈｅ　ＥＧＦＲ　ａｔ　ｔｈｅ　ｅｎｄ　ｏｆ　ｔｈｅ　ｃａｐａｃｉｔａｔｉｏｎ　ｅｎｈａｎｃｅｓ　ｉｎｔｒａｃｅｌｌｕｌａｒ　Ｃａ　ｃｏｎｃｅｎｔｒａｔｉｏｎ　ｌｅａｄｉｎｇ　ｔｏ　Ｆ－ａｃｔｉｎ　ｂｒｅａｋｄｏｗｎ　ａｎｄ　ａｌｌｏｗｓ　ｔｈｅ　ＡＲ　ｔｏ　ｔａｋｅ　ｐｌａｃｅ．Ｕｎｄｅｒ／ｎ　ｖｉｖｏ　ｃｏｎｄｉｔｉｏｎｓ．ｔｈｅ　ＥＧＦＲ　ｃａｎ　ｂｅ　ｄｉｒｅｃｔｌｙ　ａｃｔｉｖａｔｅｄ　ｂｙ　ｉｔｓ　ｋｎｏｗｎ　ｌｉｇａｎｄ　ｅｐｉｄｅｒｍａｌ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ（ＥＧＦ）．ａｎｄ　ｉｎｄｉｒｅｃｔｌｙ　ｂｙ　ａｃｔｉｖａｔｉｎｇ　ＰＫＡ　ｏｒ　ｂｙ　ｔｒａｎｓａｃｔｉｖａｔｉｏｎ　ｍｅｄｉａｔｅｄ　ｂｙ　Ｇ　ｐｒｏｔｅｉｎ．ｃｏｕｐｌｅｄ　ｒｅｃｅｐｔｏｒｓ　（ＧＰＣＲｓ）ａｃｔｉｖａｔｉｏｎ　ｏｒ　ｂｙ　ｏｕａｂａｉｎ．Ｕｎｄｅｒ　ｐｈｙｓｉｏｌｏｇｉｃａｌ　ｃｏｎｄｉｔｉｏｎｓ，ｓｐｅｒｍ　ＰＫＡ　ｉｓ　ａｃｔｉｖａｔｅｄ　ｍａｉｎｌｙ　ｂｙ　ｂｉｃａｒｂｏｎａｔｅ，ｗｈｉｃｈ　ａｃｔｉｖａｔｅｓ　ｔｈｅ　ｓｏｌｕｂｌｅ　ａｄｅｎｙｌｙｌ　ｃｙｃｌａｓｅ　ｔｏ　ｐｒｏｄｕｃｅ　ｃｙｃｌｉｃ　ａｄｅｎｏｓｉｎｅ　ｍｏｎｏｐｈｏｓｐｈａｔｅ（ｃＡＭＰ）．ｔｈｅ　ａｃｔｉｖａｔｏｒ　ｏｆ　ＰＫＡ。Ｔｈｅ　ＧＰＣＲ　ａｃｔｉｖａｔｏｒｓ　ａｎｇｉｏｔｅｎｓｉｎ　ｌｌ　ｏｒ　ｌｙｓｏｐｈｏｓｐｈａｔｉｄｉｃ　ａｃｉｄ。ａｓ　ｗｅｌＩ　ａｓ　ｏｕａｂａｉｎ　ａｎｄ　ＥＧＦ　ａｒｅ　ｐｈｙｓｉｏｌｏｇｉｃａｌ　ｃｏｒｎｐｏｎｅｎｔｓ　ｐｒｅｓｅｎｔ　ｉｎ　ｔｈｅ　ｆｅｍａｌｅ　ｒｅｐｒｏｄｕｃｔｉｖｅ　ｔｒａｃｔ．　Ａｓｉａｎ　Ｊｏｕｒｎａｆ　ｏｆＡｎｄｒｏｌｏｇｙ（２０１１）１３，１Ｏ６卜ＩＩ１０；ｄｏｉ：１０．１０３８／ａｊａ．２０１０．７８：ｐｕｂｌｉｓｈｅｄ　ｏｎｌｉｎｅ　１８　Ｏｃｔｏｈｅｒ　２Ｏ１０　

Ｋｅｙｗｏｒｄｓ：ａｃｒｏｓｏｍｅ　ｒｅａｃｔｉｏｎ；ｃａｐａｃｉｔａｔｉｏｎ；ＰＩ３Ｋ：ＰＫＡ；ＰＫＣ；ｓｐｅｒｍａｔｏｚｏａ　

Ｉ　ＮＴＲＯＤＵＣＴ１０Ｎ　Ｅｊａｃｕｌａｔｅｄ　ｍａｍｍａｌｉａｎ　ｓｐｅｒｍａｔｏｚｏａ　ｓｈｏｕｌｄ　ｒｅｓｉｄｅ　ｉｎ　ｔｈｅ　ｆｅｍａｌｅ　ｇｅｎｉｔａｌ　ｔｒａｃｔ　ｆｏｒ　ｓｅｖｅｒａｌ　ｈｏｕｒｓ　ｂｅｆｏｒｅ　ｇａｉｎｉｎｇ　ｔｈｅ　ａｂｉｌｉｔｙ　ｔｏ　ｆｅｒｔｉｌｉｚｅ　ｔｈｅ　ｅｇｇ．Ｉｎ　ｈｕｍａｎ　ｓｐｅｒｍ，ｈｏｗｅｖｅｒ，ｓｐｅｒｍ　ｍｕｓｔ　ｍｏｖｅ　ｏｕｔ　ｏｆ　ｔｈｅ　ｓｅｍｉｎａｌ　ｐｌａｓｍａ　ｉｍｍｅｄｉａｔｅｌｙ　ａｆｔｅｒ　ｅｊａｃｕｌａｔｉｏｎ　ａｎｄ　ａｐｐｅａｒ　ｉｎ　ｔｈｅ　ｆａｌｌｏｐｉａｎ　ｔｕｂｅ　ｗｉｔｈｉｎ　ｍｉｎｕｔｅｓ．Ａｓ　ｓｏｏｎ　ａｓ　ｓｐｅｒｍ　ａｒｅ　ｍｏｖｉｎｇ　ｏｕｔ　ｏｆ　ｔｈｅ　ｅｊａｃｕｌａｔｅ　ａｎｄ　ｐａｓｓｉｎｇ　ｔｈｅ　ｃｅｒｖｉｃａｌ　ｍｕｃｕｓ，ｔｌ１ｅｖ　ｕｎｄｅｒｇｏ　ｓｅｖｅｒａｌ　ｂｉｏｃｈｅｍｉｃａｌ　ｃｈａｎｇｅｓ　ｃｏｌｌｅｃｔｉｖｅｌｙ　ｃａｌｌｅｄ　ｃａｐａｃｉｔａｔｉｏｎ（ｒｅｎｅｗｅｄ　ｉｎ　Ｆｌｏｒｍａｎ　ａｎｄ　Ｄｕｄｂｅｌｌａ　ａｎｄ　Ｇａｄｅｌｌａ　ａｎｄ　Ｖｉｓｃｏｎｔｉ　）．Ｔｈｅｓｅ　ｃｈａｎｇｅｓ　ａｒｅ　ｓｔｉｌｌ　ｎｏｔ　ｃｌｅａｒ，ｂｕｔ　ｉｔ　ｓｅｅｍｓ　ｃｅｒｔａｉｎ　ｔｏ　ｉｎｖｏｌｖｅ　ｍｏｌｅｃｕｌｅｓ　ａｂｓｏｒｂｅｄ　ｏｎ，ｏｒ　ｉｎｔｅｇｒａｔｅｄ　ｉｎｔｏ，ｔｈｅ　ｓｐｅｒｍ　ｐｌａｓｍａ　ｍｅｍｂｒａｎｅ　ｄｕｒｉｎｇ　ｅｐｉｄｉｄｙｒｎａｌ　ｍａｍｒａｔｉｏｎ，ａｎｄ　ｏｎ　ｃｏｎｔａｃｔ　ｏｆ　ｓｐｅｒｍａｔｏｚｏａ　ｗｉｔｈ　ｔｈｅ　ｓｅｍｉｎａｌ　ｐｌａｓｍａ　ｔｈｅｙ　ｒｅｎｄｅｒ　ｔｈｅ　ｓｐｅｒｍａｔｏｚｏａ　ｃａｐａｂｌｅ　ｏｆｆｅｒｔｉｌｉｚａｔｉｏｎ．Ｔｈｅ　ｒｅｍｏｖａｌ　ｏｒ　ａｌｔｅｒａｔｉｏｎ　ｏｆ　ｔｌｌｅｓｅ　ｍｏｌｅｃｕｌｅｓ　ｐｒｅｐａｒｅｓ　ｔｈｅ　ｓｐｅｒｍ　ｔｏｗａｒｄ　ｓｕｃｃｅｓｓＥｄ　ｂｉｎｄｉｎｇ　ｔｏ　ｔｈｅ　ｅｇｇ　ａｎｄ　ｆｅｒｔｉｌｉｚａｔｉｏｎ．Ｉｔ　ｗａｓ　ｓｈｏｗｎ　ｂｙ　Ｙａｎａｇｉｍａｃｈｉ　ａｎｄ　Ｃｈａｎｇ３　ｔｈａｔ　ｃａｐａｃｉｔａｔｉｏｎ　ｃａｎ　ｂｅ　ｍｉｍｉｃｋｅｄ　ｉｎ　ｖｉｔｒｏ，　ｍａｋｉｎｇ　ｔｈｅ　ａｎａｌｙｓｉｓ　ｏｆ　ｃａｐａｃｉｔａｔｉｏｎ　ｍｅｃｈａｎｉｓｍｓ　ｃｏｎｓｉｄｅｒａｂｌｙ　ｅａｓｉｅｒ．　Ｄｕｒｉｎｇ　ｍａｍｍａｌｉａｎ　ｆｅｒｔｉｌｉｚａｔｉｏｎ，ｔｈｅ　ｃａｐａｃｉｔａｔｅｄ　ｓｐｅｒｍａｔｏｚｏｏｎ　ｐｅｎｅｔｒａｔｅｓ　ｔｈｅ　ｃｕｍｕｌｕｓ　ｏｏｐｈｒｏｕｓ　ｏｆ　ｔｈｅ　ｏｖｕｍ，ａｎｄ　ｔｈｅｎ　ｂｉｎｄｓ　ｔｏ　ｔｈｅ　ｚｏｎａ　ｐｅｌｌｕｃｉｄａ　ｗｉｔｈ　ｉｔｓ　ｐｌａｓｍａ　ｍｅｍｂｒａｎｅ　ｉｎｔａｃｔ．Ａｆｔｅｒ　ｂｉｎｄｉｎｇ　ｔｏ　ｔｈｅ。ｇｇ　ｚｏｎａ　ｐｅｌｌｕｃｉｄａ，ｔｈｅ　ｓｐｅｒｍａｔｏｚｏｏｎ　ｕｎｄｅｒｇｏｅｓ　ａｎ　ｅｘｏｃｙｔｏｔｉｃ　ｐｒｏｃｅｓｓ　ｃａｌｌｅｄ　ｔｈｅ　ａｃｒｏｓｏｍｅ　ｒｅａｃｔｉｏｎ（ＡＲ）（ｒｅｖｉｅｗｅｄ　ｉｎ　Ｙａｎａｇｉｍａｃｈｉ　ａ１．，　Ｒｏｌｄａｎ　ａｎｄ　Ｓｈｉ，’Ｆｌｏｒｍａｎ　ｅｔ　ａ１．。ａｎｄ　Ｂｒｅｉｔｂａｒｔ，１．Ｔｈｉｓ　ｅｖｅｎｔ　ｉｓ　ｒｅｑｕｉｒｅｄ　ｆｏｒ　ｆｅｒｔｉｌｉｚａｔｉｏｎ，ｂｅｃａｕｓｅ　ｉｔ　ｅｎａｂｌｅｓ　ｐａｓｓａｇｅ　ｏｆ　ｔｈｅ　ｓｐｅｒｍａｔｏ—　ｚｏｏｎ　ｔｈｒｏｕｇｈ　ｔｈｅ　ｚｏｎａ　ｐｅｌｌｕｃｉｄａ　ａｎｄ　ｉｔｓ　ｓｕｂｓｅｑｕｅｎｔ　ｆｕｓｉｏｎ　ｗｉｔｈ　ｔｈｅ　ｅｇｇ　ｏｏｌｅｍａ．Ｔｈｅｒｅｆｏｒｅ，ｅｌｕｃｉｄａｔｉｏｎ　ｏｆ　ｔｈｅ　ｍｅｃｈａｎｉｓｍｓ　ｒｅｇｕｌａｔｉｎｇ　ｔｈｅ　ＡＲ　ｉｓ　ｉｍｐｏｒｔａｎｔ　ｆｏｒ　ｕｎｄｅｒｓｔａｎｄｉｎｇ　ｔｈｅ　ｐｒｏｃｅｓｓ　ｏｆ　ｍａｍｍａｌｉａｎ　ｆｅｒｔ—　ｉｌｉｚａｔｉｏｎ．Ｉｎ　ｏｕｒ　ｌａｂｏｒａｔｏｒｙ　ａｎｄ　ｏｔｈｅｒｓ　ｉｔ　ｗａｓ　ｓｈｏｗｎ　ｔｈａｔ　ｖａｒｉｅｔｙ　ｏｆ　ａｇｏｎｉｓｔｓ　ｃａｎ　ｔｒｉｇｇｅｒ　ｔｈｅ　ＡＲ　ｖｉａ　ｒｅｃｅｐｔｏｒ—ｍｅｄｉａｔｅｄ　ｍｅｃｈａｎｉｓｍｓ．　’　一　”　Ｍｔｈｏｕｇｈ　ｚｏｎａ　ｐｅｌｌｕｃｉｄａ—ｄｅｒｉｖｅｄ　ｇｌｙｃｏｐｒｏｔｅｉｎｓ　ａｒｅ　ｔｈｏｕｇｈｔ　ｔｏ　ｂｅ　ｔｈｅ　ｐｈｙｓｉｏｌｏｇｉｃａｌ　ｉｎｄｕｃｅｒｓ　ｏｆ　ｔｈｅ　ＡＲ，　１，１２　ｔｈｅ　ｒｅａｃｔｉｏｎ　ｃａｎ　ｂｅ　ｉｎｄｕｃｅｄ　

ｖｉｔｒｏ　ｂｙ　ｖａｒｉｏｕｓ　ｃｏｎｓｔｉｔｕｅｎｔｓ　ｏｆｔｈｅ　ｆｅｍａｌｅ　ｒｅｐｒｏｄｕｃｔｉｖｅ　ｔｒａｃｔ　ｉｎｃｌｕｄｉｎｇ　ｐｒｏｇｅｓｔｅｒｏｎｅ，　。’　ｐｒｏｓｔａｇｌａｎｄｉｎｓ，　ａｔｒｉａｌ　ｎａｔｒｉｕｒｅｔｉｃ　ｐｅｐｔｉｄｅ，　ｅｐｉ．　

ｄｅｒｍａｌ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ（ＥＧＦ），　，　。，　ｏｕａｂａｉｎ　ａｎｄ　ｏｔｈｅｒ　ｌｉｇａｎｄｓ．　

Ｔｈｅｓｅ　ａｇｏｎｉｓｔｓ　ｍａｙ　ｈａｖｅ　ａ　ｄｉｒｅｃｔ　ａｎｄ／ｏｒ　ｓｙｎｅｒｇｉｓｔｉｃ　ｅｆｆｅｃｔ　ｗｉｔｈ　ｏｔｈｅｒ　ｃｏｎｓｔｉｔｕｅｎｔｓ　ｏｆ　ｔｈｅ　ｆｅｍａｌｅ　ｒｅｐｒｏｄｕｃｔｉｖｅ　ｏｒ　ｏｎ　ｔｈｅ　ｚｏｎａ　ｐｅｌｌｕｃｉｄａ．１４　

Ｔｈｅ　ｑｕｅｓｔｉｏｎ　ｒｅｇａｒｄｉｎｇ　ｔｈｅ　ｐｈｙｓｉｏｌｏｇｉｃａｌ　ｒｏｌｅ　ｏｆｔｈｅｓｅ　ｆａｃｔｏｒｓ　ｕｎｄｅｒ　ｉｎ　ｖｉｖｏ　ｃｏｎｄｉｔｉｏｎｓ　ｉｓ　ｓｔｉｌｌ　ａｎ　ｏｐｅｎ　ｑｕｅｓｔｉｏｎ．Ａｓｓｕｍｉｎｇ　ｔｈａｔ　ａｃｒｏｓｏｍｅ—　ｒｅａｃｔｅｄ　ｓｐｅｒｍ　ｃａｎｎｏｔ　ｂｉｎｄ　ａｎｄ　ｆｅｒｔｉｌｉｚｅ　ｔｈｅ　ｅｇｇ，ｗｅ　ｓｕｇｇｅｓｔ　ｔｈａｔ　ｐｒｅ—　ｍａｔｕｒｅ　ＡＲ　ｂｅｆｏｒｅ　ｒｅａｃｈｉｎｇ　ｔｈｅ　ｅｇｇ　ｚｏｎａ　ｐｅｌｌｕｃｉｄａ，ｍｉｇｈｔ　ｂｅ　ａ　ｗａｙ　ｏｆ　ｓｅｌｅｃｔｉｏｎ　ｉｎ　ｗｈｉｃｈ　ｔｈｅ‘ｂａｄ’ｓｐｅｒｍ　ｗｉｌｌ　ｕｎｄｅｒｇｏ　ｔｈｅ　ｓｏ—ｃａｌｌｅｄ　ｎｏｎ．　ｓｐｅｃｉｆｉｃ　ＡＲ　ａｎｄ　ｗｏｕｌｄ　ｎｏｔ　ｂｅ　ａｂｌｅ　ｔｏ　ｆｅｒｔｉｌｉｚｅ，ｗｈｅｒｅａｓ　ｔｈｅ‘ｂｅｓｔ’　ｓｅｌｅｃｔｅｄ　ｓｐｅｒｍ　ｗｉｌｌ　ｒｅａｃｈ　ｔｈｅ　ｅｇｇ　ｉｎ　ｉｔｓ　ｉｎｔａｃｔ　ｍｏｒｐｈｏｌｏｇｙ　ａｎｄ　ｗｉｌｌ　ｆｅｒｔｉｌｉｚｅ　ｉｔ．Ｔｈｕｓ，ｔｏ　ｓｔｕｄｙ　ｔｈｅ　ｓｅｌｅｃｔｉｏｎ　ｍｅｃｈａｎｉｓｍ，ｉｔ　ｉｓ　ｖｅｒｙ　ｉｍｐｏｒｔａｎｔ　ｔｏ　ｕｎｄｅｒｓｔａｎｄ　ｔｈｅ　ｍｅｃｈａｎｉｓｍ　ｏｆ　ａｃｔｉｏｎ　ｏｆ　ｔｈｅ　ｖａｒｉｏｕｓ　ｐｈｙｓｉｏｌｏｇｉｃａｌ　ｆａｃｔｏｒｓ　ｔｈａｔ　ｉｎｄｕｃｅ　ｔｈｅ　ＡＲ．Ｏｎｅ　ｏｆｔｈｅｓｅ　ｍｅｃｈａｎｉｓｍｓ，ｔｈｅ　ＥＧＦ　ｒｅｃｅｐｔｏｒ　（ＥＧＦＲ）ｓｙｓｔｅｍ　ｉｓ　ｄｅｓｃｒｉｂｅｄ　ｉｎ　ｔｈｉｓ　ｒｅｖｉｅｗ．　