Hedgehog配体释放过程中的关键蛋白Disp1和Scube2的结构与功能研究

Hedgehog（Hh）信号通路是一条参与调控胚胎发育和组织稳态的重要信号转导通路，其失调会引起先天性发育畸形以及多种相关癌症。Hh配体是一种独特的具有双重脂修饰的蛋白质，包括氨基端的棕榈酰修饰和羧基端的胆固醇修饰。这种双重脂质修饰使Hh配体具有极强的疏水性，使其牢固地附着在产生Hh细胞的质膜上。然而，在发育过程中，Hh配体需要从生产细胞中释放并扩散向靶细胞。因此，生物体发展出了一套专门的系统来帮助完成Hh配体的释放和运输。该系统主要包括细胞膜上的Dispatched（Disp）蛋白和细胞外可溶性的Scube蛋白。

Disp蛋白在发挥功能前，需要Furin蛋白酶在其第一个胞外区结构域（ECD1）进行剪切。然而，该剪切过程如何激活Disp蛋白，激活后的Disp蛋白如何识别Hh配体以及后续配体的转移释放等分子机制目前均不清楚。本研究通过解析了不同状态的Disp蛋白以及和Hh配体（Shh）复合物的多个高分辨率冷冻电镜结构，结合生物化学和细胞生物学实验，清楚地阐释了Disp蛋白的剪切-激活机制和配体释放的分子机制。首先，创造性地通过将Disp1中被Furin识别的区域替换为具有高度特异性的3C蛋白酶的裂解位点来捕获剪切前后的Disp1蛋白，通过pull down实验，证明了Disp1的剪切加工可以调控与Hh配体的相互作用，并在此基础上成功解析了Disp1蛋白两种状态下的冷冻电镜结构，阐明了Disp1蛋白剪切-激活的分子机制。剪切过程移除了由Furin酶切位点构成的空间位阻，并使得Disp1蛋白的胞外区结构域（ECD1和ECD2）之间更为开放，从而大大增强了Disp1与Hh配体的结合能力。其次，解析了Disp1与Shh的复合物结构，揭示了哺乳动物与果蝇的Disp与Hh配体在结合模式上存在差异，提出Hh的释放机制在物种间并不是严格保守的。最后，通过生化实验证明了Scube2与Shh能够形成稳定的二元复合物，并且Shh的胆固醇修饰对于该复合物的稳定性起重要作用。

综合以上数据，提出了Disp1在Hh配体释放过程中的工作机制。Disp1首先通过蛋白剪切机制引起构象的变化，增强了与Shh的结合能力；当与配体Shh结合后，Disp1进一步发生构象变化，向内旋转并牢牢的抓住Shh；接着将Shh传递给Scube蛋白；最后，由Scube蛋白作为伴侣结合Hh配体，协助其从生产细胞运输到靶细胞。

The Hedgehog (Hh) signaling pathway is an essential signaling for animal development and tissue homeostasis, and its dysregulation can lead to congenital developmental abnormalities and various cancers. The Hh ligand is a unique protein with dual lipid modifications, including palmitoylation at the amino terminus and cholesterol modification at the carboxyl terminus. This dual lipid modification confers strong hydrophobicity to the Hh ligand, facilitating its stable attachment to the plasma membrane of producing cells. However, during development, the Hh ligand needs to be released from producing cells and spread to target cells. Therefore, organisms have evolved a specialized system to assist in the release and transport of the Hh ligand, primarily involving the transmembrane protein Dispatched (Disp) and the secreted protein Scube.

Disp function requires Furin-mediated proteolytic cleavage of its extracellular domain (ECD1). However, the molecular mechanisms underlying how this cleavage activates Disp protein, how activated Disp protein recognizes the Hh ligand, and the subsequent transfer and release of the ligand remain unclear. This study elucidates the cleavage-activation mechanism of Disp protein and the molecular mechanism of ligand release by analyzing multiple high-resolution cryo-electron microscopy structures of Disp protein in different states and its complex with the Hh ligand (Shh), combined with biochemical and cell biology experiments. Firstly, we creatively captured the pre- and post-cleavage states of Disp1 protein by replacing the region recognized by Furin with a cleavage site of highly specific 3C protease. Through pull-down assay, we demonstrated that cleavage processing of Disp1 regulates its interaction with Hh ligands. Based on this, we successfully resolving the cryo-EM structures of Disp1 protein in two states, elucidating the molecular mechanism of cleavage-activation of Disp1 protein. Cleavage removes the steric hindrance formed by the Furin cleavage site, leading to a more open conformation between the extracellular domains (ECD1 and ECD2) of Disp1 protein, significantly enhancing its binding affinity with the Hh ligand. Secondly, we elucidated the structure of the Disp1-Shh complex and revealed differences in the interaction between mammalian and Drosophila in Disp and Hh ligand interaction, suggesting that the Hh release mechanism is not strictly conserved across species. We demonstrated through biochemical experiments that Scube2 can form a stable binary complex with Shh, with the cholesterol modification of Shh playing a crucial role in the stability of this complex.

Based on the above data, we propose the working mechanism of Disp1 in the process of Hh ligand release. Disp1 first undergoes conformational changes through protein cleavage, enhancing its binding ability to Shh; when bound to the Shh ligand, Disp1 further undergoes conformational changes, firmly capturing Shh; subsequently, Disp1 transfers Shh to the Scube protein; finally, Scube protein, as a partner, binds to the Hh ligand, assisting its transport from producing cells to target cells.

[1] INGHAM P W, MCMAHON A P. Hedgehog signaling in animal development: paradigms and principles[J]. Genes Dev, 2001, 15(23): 3059-3087.
[2] PETROV K, WIERBOWSKI B M, SALIC A. Sending and receiving hedgehog signals[J]. Annu Rev Cell Dev Biol, 2017, 33: 145-168.
[3] HILL P, WANG B, RUTHER U. The molecular basis of Pallister Hall associated polydactyly[J]. Hum Mol Genet, 2007, 16(17): 2089-2096.
[4] GENG X, OLIVER G. Pathogenesis of holoprosencephaly[J]. J Clin Invest, 2009, 119(6): 1403-1413.
[5] JIANG J, HUI C C. Hedgehog signaling in development and cancer[J]. Dev Cell, 2008, 15(6): 801-812.
[6] PAK E, SEGAL R A. Hedgehog signal transduction: key players, oncogenic drivers, and cancer therapy[J]. Dev Cell, 2016, 38(4): 333-344.
[7] BARAKAT M T, HUMKE E W, SCOTT M P. Learning from Jekyll to control Hyde: Hedgehog signaling in development and cancer[J]. Trends Mol Med, 2010, 16(8): 337-348.
[8] PORTER J A, YOUNG K E, BEACHY P A. Cholesterol modification of hedgehog signaling proteins in animal development[J]. Science, 1996, 274(5285): 255-259.
[9] PEPINSKY R B, ZENG C, WEN D, et al. Identification of a palmitic acid-modified form of human Sonic hedgehog[J]. J Biol Chem, 1998, 273(22): 14037-14045.
[10] CHAMOUN Z, MANN R K, NELLEN D, et al. Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal[J]. Science, 2001, 293(5537): 2080-2084.
[11] BURKE R, NELLEN D, BELLOTTO M, et al. Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells[J]. Cell, 1999, 99(7): 803-815.
[12] MA Y, ERKNER A, GONG R, et al. Hedgehog-mediated patterning of the mammalian embryo requires transporter-like function of dispatched[J]. Cell, 2002, 111(1): 63-75.
[13] TAIPALE J, COOPER M K, MAITI T, et al. Patched acts catalytically to suppress the activity of Smoothened[J]. Nature, 2002, 418(6900): 892-897.
[14] COHEN D J. Targeting the hedgehog pathway: role in cancer and clinical implications of its inhibition[J]. Hematol Oncol Clin North Am, 2012, 26(3): 565-588, viii.
[15] FAN C W, TULADHAR R, LUM L. Signaling: making a leaner Hedgehog[J]. Nat Chem Biol, 2013, 9(4): 217-218.
[16] QI X, LI X. Mechanistic insights into the generation and transduction of hedgehog signaling[J]. Trends Biochem Sci, 2020, 45(5): 397-410.
[17] NUSSLEIN-VOLHARD C, WIESCHAUS E. Mutations affecting segment number and polarity in Drosophila[J]. Nature, 1980, 287(5785): 795-801.
[18] LEE J J, VON KESSLER D P, PARKS S, et al. Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog[J]. Cell, 1992, 71(1): 33-50.
[19] INOUE Y, NIWA N, MITO T, et al. Expression patterns of hedgehog, wingless, and decapentaplegic during gut formation of Gryllus bimaculatus (cricket)[J]. Mech Dev, 2002, 110(1-2): 245-248.
[20] KUWABARA P E, LEE M H, SCHEDL T, et al. A C. elegans patched gene, ptc-1, functions in germ-line cytokinesis[J]. Genes Dev, 2000, 14(15): 1933-1944.
[21] VARJOSALO M, TAIPALE J. Hedgehog: functions and mechanisms[J]. Genes Dev, 2008, 22(18): 2454-2472.
[22] ECHELARD Y, EPSTEIN D J, ST-JACQUES B, et al. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity[J]. Cell, 1993, 75(7): 1417-1430.
[23] KRAUSS S, CONCORDET J P, INGHAM P W. A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos[J]. Cell, 1993, 75(7): 1431-1444.
[24] RIDDLE R D, JOHNSON R L, LAUFER E, et al. Sonic hedgehog mediates the polarizing activity of the ZPA[J]. Cell, 1993, 75(7): 1401-1416.
[25] ROELINK H, AUGSBURGER A, HEEMSKERK J, et al. Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord[J]. Cell, 1994, 76(4): 761-775.
[26] MARIGO V, ROBERTS D J, LEE S M, et al. Cloning, expression, and chromosomal location of SHH and IHH: two human homologues of the Drosophila segment polarity gene hedgehog[J]. Genomics, 1995, 28(1): 44-51.
[27] EKKER S C, UNGAR A R, GREENSTEIN P, et al. Patterning activities of vertebrate hedgehog proteins in the developing eye and brain[J]. Curr Biol, 1995, 5(8): 944-955.
[28] CURRIE P D, INGHAM P W. Induction of a specific muscle cell type by a hedgehog-like protein in zebrafish[J]. Nature, 1996, 382(6590): 452-455.
[29] MCMAHON A P, INGHAM P W, TABIN C J. Developmental roles and clinical significance of hedgehog signaling[J]. Curr Top Dev Biol, 2003, 53: 1-114.
[30] BITGOOD M J, SHEN L, MCMAHON A P. Sertoli cell signaling by Desert hedgehog regulates the male germline[J]. Curr Biol, 1996, 6(3): 298-304.
[31] COLNOT C, DE LA FUENTE L, HUANG S, et al. Indian hedgehog synchronizes skeletal angiogenesis and perichondrial maturation with cartilage development[J]. Development, 2005, 132(5): 1057-1067.
[32] HELLEMANS J, COUCKE P J, GIEDION A, et al. Homozygous mutations in IHH cause acrocapitofemoral dysplasia, an autosomal recessive disorder with cone-shaped epiphyses in hands and hips[J]. Am J Hum Genet, 2003, 72(4): 1040-1046.
[33] PAGAN-WESTPHAL S M, TABIN C J. The transfer of left-right positional information during chick embryogenesis[J]. Cell, 1998, 93(1): 25-35.
[34] CHIANG C, LITINGTUNG Y, LEE E, et al. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function[J]. Nature, 1996, 383(6599): 407-413.
[35] ZHANG Y, KALDERON D. Hedgehog acts as a somatic stem cell factor in the Drosophila ovary[J]. Nature, 2001, 410(6828): 599-604.
[36] BUGLINO J A, RESH M D. Hhat is a palmitoylacyltransferase with specificity for N-palmitoylation of Sonic Hedgehog[J]. J Biol Chem, 2008, 283(32): 22076-22088.
[37] CHEN M H, LI Y J, KAWAKAMI T, et al. Palmitoylation is required for the production of a soluble multimeric Hedgehog protein complex and long-range signaling in vertebrates[J]. Genes Dev, 2004, 18(6): 641-659.
[38] CREANGA A, GLENN T D, MANN R K, et al. Scube/You activity mediates release of dually lipid-modified Hedgehog signal in soluble form[J]. Genes Dev, 2012, 26(12): 1312-1325.
[39] MANN R K, BEACHY P A. Cholesterol modification of proteins[J]. Biochim Biophys Acta, 2000, 1529(1-3): 188-202.
[40] JIANG Y, BENZ T L, LONG S B. Substrate and product complexes reveal mechanisms of Hedgehog acylation by HHAT[J]. Science, 2021, 372(6547): 1215-1219.
[41] HALL T M, PORTER J A, YOUNG K E, et al. Crystal structure of a Hedgehog autoprocessing domain: homology between Hedgehog and self-splicing proteins[J]. Cell, 1997, 91(1): 85-97.
[42] CHEN X, TUKACHINSKY H, HUANG C H, et al. Processing and turnover of the Hedgehog protein in the endoplasmic reticulum[J]. J Cell Biol, 2011, 192(5): 825-838.
[43] WILKINSON B, GILBERT H F. Protein disulfide isomerase[J]. Biochim Biophys Acta, 2004, 1699(1-2): 35-44.
[44] PUROHIT R, PENG D S, VIELMAS E, et al. Dual roles of the sterol recognition region in Hedgehog protein modification[J]. Commun Biol, 2020, 3(1): 250.
[45] MAFI A, PUROHIT R, VIELMAS E, et al. Hedgehog proteins create a dynamic cholesterol interface[J]. PLoS One, 2021, 16(2): e0246814.
[46] TANG X, CHEN R, MESIAS V S D, et al. A SURF4-to-proteoglycan relay mechanism that mediates the sorting and secretion of a tagged variant of sonic hedgehog[J]. Proc Natl Acad Sci U S A, 2022, 119(11): e2113991119.
[47] GALLET A, RODRIGUEZ R, RUEL L, et al. Cholesterol modification of hedgehog is required for trafficking and movement, revealing an asymmetric cellular response to hedgehog[J]. Dev Cell, 2003, 4(2): 191-204.
[48] GUERRERO I, CHIANG C. A conserved mechanism of Hedgehog gradient formation by lipid modifications[J]. Trends Cell Biol, 2007, 17(1): 1-5.
[49] LI Y, ZHANG H, LITINGTUNG Y, et al. Cholesterol modification restricts the spread of Shh gradient in the limb bud[J]. Proc Natl Acad Sci U S A, 2006, 103(17): 6548-6553.
[50] DESSAUD E, MCMAHON A P, BRISCOE J. Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network[J]. Development, 2008, 135(15): 2489-2503.
[51] TICKLE C, TOWERS M. Sonic hedgehog signaling in limb development[J]. Front Cell Dev Biol, 2017, 5: 14.
[52] HARFE B D, SCHERZ P J, NISSIM S, et al. Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities[J]. Cell, 2004, 118(4): 517-528.
[53] HALL E T, CLEVERDON E R, OGDEN S K. Dispatching Sonic Hedgehog: molecular mechanisms controlling deployment[J]. Trends Cell Biol, 2019, 29(5): 385-395.
[54] COULTER M E, DOROBANTU C M, LODEWIJK G A, et al. The ESCRT-III protein CHMP1A mediates secretion of Sonic Hedgehog on a distinctive subtype of extracellular vesicles[J]. Cell Rep, 2018, 24(4): 973-986 e978.
[55] GRADILLA A C, GONZALEZ E, SEIJO I, et al. Exosomes as Hedgehog carriers in cytoneme-mediated transport and secretion[J]. Nat Commun, 2014, 5: 5649.
[56] MATUSEK T, WENDLER F, POLES S, et al. The ESCRT machinery regulates the secretion and long-range activity of Hedgehog[J]. Nature, 2014, 516(7529): 99-103.
[57] GALLET A, RUEL L, STACCINI-LAVENANT L, et al. Cholesterol modification is necessary for controlled planar long-range activity of Hedgehog in Drosophila epithelia[J]. Development, 2006, 133(3): 407-418.
[58] ZENG X, GOETZ J A, SUBER L M, et al. A freely diffusible form of Sonic hedgehog mediates long-range signalling[J]. Nature, 2001, 411(6838): 716-720.
[59] VYAS N, GOSWAMI D, MANONMANI A, et al. Nanoscale organization of hedgehog is essential for long-range signaling[J]. Cell, 2008, 133(7): 1214-1227.
[60] PANAKOVA D, SPRONG H, MAROIS E, et al. Lipoprotein particles are required for Hedgehog and Wingless signalling[J]. Nature, 2005, 435(7038): 58-65.
[61] PALM W, SWIERCZYNSKA M M, KUMARI V, et al. Secretion and signaling activities of lipoprotein-associated hedgehog and non-sterol-modified hedgehog in flies and mammals[J]. PLoS Biol, 2013, 11(3): e1001505.
[62] CALLEJO A, BILIONI A, MOLLICA E, et al. Dispatched mediates Hedgehog basolateral release to form the long-range morphogenetic gradient in the Drosophila wing disk epithelium[J]. Proc Natl Acad Sci U S A, 2011, 108(31): 12591-12598.
[63] SANDERS T A, LLAGOSTERA E, BARNA M. Specialized filopodia direct long-range transport of SHH during vertebrate tissue patterning[J]. Nature, 2013, 497(7451): 628-632.
[64] GONZALEZ-MENDEZ L, SEIJO-BARANDIARAN I, GUERRERO I. Cytoneme-mediated cell-cell contacts for Hedgehog reception[J]. Elife, 2017, 6.
[65] BODEEN W J, MARADA S, TRUONG A, et al. A fixation method to preserve cultured cell cytonemes facilitates mechanistic interrogation of morphogen transport[J]. Development, 2017, 144(19): 3612-3624.
[66] ROY S, HUANG H, LIU S, et al. Cytoneme-mediated contact-dependent transport of the Drosophila decapentaplegic signaling protein[J]. Science, 2014, 343(6173): 1244624.
[67] D'ANGELO G, MATUSEK T, PIZETTE S, et al. Endocytosis of Hedgehog through dispatched regulates long-range signaling[J]. Dev Cell, 2015, 32(3): 290-303.
[68] YANG T, ESPENSHADE P J, WRIGHT M E, et al. Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER[J]. Cell, 2002, 110(4): 489-500.
[69] BROWN M S, RADHAKRISHNAN A, GOLDSTEIN J L. Retrospective on cholesterol homeostasis: the central role of Scap[J]. Annu Rev Biochem, 2018, 87: 783-807.
[70] VYAS N, WALVEKAR A, TATE D, et al. Vertebrate Hedgehog is secreted on two types of extracellular vesicles with different signaling properties[J]. Sci Rep, 2014, 4: 7357.
[71] ETHERIDGE L A, CRAWFORD T Q, ZHANG S, et al. Evidence for a role of vertebrate Disp1 in long-range Shh signaling[J]. Development, 2010, 137(1): 133-140.
[72] QI X, SCHMIEGE P, COUTAVAS E, et al. Structures of human Patched and its complex with native palmitoylated sonic hedgehog[J]. Nature, 2018, 560(7716): 128-132.
[73] STEWART D P, MARADA S, BODEEN W J, et al. Cleavage activates dispatched for Sonic Hedgehog ligand release[J]. Elife, 2018, 7.
[74] SKODA A M, SIMOVIC D, KARIN V, et al. The role of the Hedgehog signaling pathway in cancer: A comprehensive review[J]. Bosn J Basic Med Sci, 2018, 18(1): 8-20.
[75] COHEN M M, JR. Hedgehog signaling update[J]. Am J Med Genet A, 2010, 152A(8): 1875-1914.
[76] KAUVAR E F, MUENKE M. Holoprosencephaly: recommendations for diagnosis and management[J]. Curr Opin Pediatr, 2010, 22(6): 687-695.
[77] MOUDEN C, DUBOURG C, CARRE W, et al. Complex mode of inheritance in holoprosencephaly revealed by whole exome sequencing[J]. Clin Genet, 2016, 89(6): 659-668.
[78] YAMAGUCHI A, NAKASHIMA R, SAKURAI K. Structural basis of RND-type multidrug exporters[J]. Front Microbiol, 2015, 6: 327.
[79] KUWABARA P E, LABOUESSE M. The sterol-sensing domain: multiple families, a unique role?[J]. Trends Genet, 2002, 18(4): 193-201.
[80] CANNAC F, QI C, FALSCHLUNGER J, et al. Cryo-EM structure of the Hedgehog release protein Dispatched[J]. Sci Adv, 2020, 6(16): eaay7928.
[81] CHEN H, LIU Y, LI X. Structure of human Dispatched-1 provides insights into Hedgehog ligand biogenesis[J]. Life Sci Alliance, 2020, 3(8).
[82] LUO Y, WAN G, ZHOU X, et al. Architecture of Dispatched, a transmembrane protein responsible for Hedgehog release[J]. Front Mol Biosci, 2021, 8: 701826.
[83] WANG Q, ASARNOW D E, DING K, et al. Dispatched uses Na(+) flux to power release of lipid-modified Hedgehog[J]. Nature, 2021, 599(7884): 320-324.
[84] PETROV K, WIERBOWSKI B M, LIU J, et al. Distinct cation gradients power cholesterol transport at different key points in the Hedgehog signaling pathway[J]. Dev Cell, 2020, 55(3): 314-327 e317.
[85] TUKACHINSKY H, KUZMICKAS R P, JAO C Y, et al. Dispatched and scube mediate the efficient secretion of the cholesterol-modified hedgehog ligand[J]. Cell Rep, 2012, 2(2): 308-320.
[86] WIERBOWSKI B M, PETROV K, ARAVENA L, et al. Hedgehog pathway activation requires coreceptor-catalyzed, lipid-dependent relay of the Sonic Hedgehog ligand[J]. Dev Cell, 2020, 55(4): 450-467 e458.
[87] GRIMMOND S, LARDER R, VAN HATEREN N, et al. Cloning, mapping, and expression analysis of a gene encoding a novel mammalian EGF-related protein (SCUBE1)[J]. Genomics, 2000, 70(1): 74-81.
[88] YANG R B, NG C K, WASSERMAN S M, et al. Identification of a novel family of cell-surface proteins expressed in human vascular endothelium[J]. J Biol Chem, 2002, 277(48): 46364-46373.
[89] TSAI M T, CHENG C J, LIN Y C, et al. Isolation and characterization of a secreted, cell-surface glycoprotein SCUBE2 from humans[J]. Biochem J, 2009, 422(1): 119-128.
[90] WU B T, SU Y H, TSAI M T, et al. A novel secreted, cell-surface glycoprotein containing multiple epidermal growth factor-like repeats and one CUB domain is highly expressed in primary osteoblasts and bones[J]. J Biol Chem, 2004, 279(36): 37485-37490.
[91] LIN Y C, SAHOO B K, GAU S S, et al. The biology of SCUBE[J]. J Biomed Sci, 2023, 30(1): 33.
[92] CHENG C J, LIN Y C, TSAI M T, et al. SCUBE2 suppresses breast tumor cell proliferation and confers a favorable prognosis in invasive breast cancer[J]. Cancer Res, 2009, 69(8): 3634-3641.
[93] LIN Y C, CHAO T Y, YEH C T, et al. Endothelial SCUBE2 interacts with VEGFR2 and regulates VEGF-induced angiogenesis[J]. Arterioscler Thromb Vasc Biol, 2017, 37(1): 144-155.
[94] VAN EEDEN F J, GRANATO M, SCHACH U, et al. Mutations affecting somite formation and patterning in the zebrafish, Danio rerio[J]. Development, 1996, 123: 153-164.
[95] HOLLWAY G E, MAULE J, GAUTIER P, et al. Scube2 mediates Hedgehog signalling in the zebrafish embryo[J]. Dev Biol, 2006, 294(1): 104-118.
[96] GRIMMOND S, LARDER R, VAN HATEREN N, et al. Expression of a novel mammalian epidermal growth factor-related gene during mouse neural development[J]. Mech Dev, 2001, 102(1-2): 209-211.
[97] GLISE B, MILLER C A, CROZATIER M, et al. Shifted, the Drosophila ortholog of Wnt inhibitory factor-1, controls the distribution and movement of Hedgehog[J]. Dev Cell, 2005, 8(2): 255-266.
[98] DAVIS C G. The many faces of epidermal growth factor repeats[J]. New Biol, 1990, 2(5): 410-419.
[99] PRIETO J H, SAMPOLI BENITEZ B A, MELACINI G, et al. Dynamics of the fragment of thrombomodulin containing the fourth and fifth epidermal growth factor-like domains correlate with function[J]. Biochemistry, 2005, 44(4): 1225-1233.
[100] LUCA V C, KIM B C, GE C, et al. Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity[J]. Science, 2017, 355(6331): 1320-1324.
[101] LO SURDO P, BOTTOMLEY M J, CALZETTA A, et al. Mechanistic implications for LDL receptor degradation from the PCSK9/LDLR structure at neutral pH[J]. EMBO Rep, 2011, 12(12): 1300-1305.
[102] STACEY M, CHANG G W, DAVIES J Q, et al. The epidermal growth factor-like domains of the human EMR2 receptor mediate cell attachment through chondroitin sulfate glycosaminoglycans[J]. Blood, 2003, 102(8): 2916-2924.
[103] PERIZ J, GILL A C, KNOTT V, et al. Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4[J]. Mol Biochem Parasitol, 2005, 143(2): 192-199.
[104] TU C F, YAN Y T, WU S Y, et al. Domain and functional analysis of a novel platelet-endothelial cell surface protein, SCUBE1[J]. J Biol Chem, 2008, 283(18): 12478-12488.
[105] LIN Y C, NICETA M, MUTO V, et al. SCUBE3 loss-of-function causes a recognizable recessive developmental disorder due to defective bone morphogenetic protein signaling[J]. Am J Hum Genet, 2021, 108(1): 115-133.
[106] TU C F, TSAO K C, LEE S J, et al. SCUBE3 (signal peptide-CUB-EGF domain-containing protein 3) modulates fibroblast growth factor signaling during fast muscle development[J]. J Biol Chem, 2014, 289(27): 18928-18942.
[107] MELLQUIST J L, KASTURI L, SPITALNIK S L, et al. The amino acid following an Asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency[J]. Biochemistry, 1998, 37(19): 6833-6837.
[108] LIAO W J, TSAO K C, YANG R B. Electrostatics and N-glycan-mediated membrane tethering of SCUBE1 is critical for promoting bone morphogenetic protein signalling[J]. Biochem J, 2016, 473(5): 661-672.
[109] BORK P, BECKMANN G. The CUB domain. A widespread module in developmentally regulated proteins[J]. J Mol Biol, 1993, 231(2): 539-545.
[110] GABORIAUD C, GREGORY-PAURON L, TEILLET F, et al. Structure and properties of the Ca(2+)-binding CUB domain, a widespread ligand-recognition unit involved in major biological functions[J]. Biochem J, 2011, 439(2): 185-193.
[111] GABORIAUD C, TEILLET F, GREGORY L A, et al. Assembly of C1 and the MBL- and ficolin-MASP complexes: structural insights[J]. Immunobiology, 2007, 212(4-5): 279-288.
[112] ANDERSEN C B, MADSEN M, STORM T, et al. Structural basis for receptor recognition of vitamin-B(12)-intrinsic factor complexes[J]. Nature, 2010, 464(7287): 445-448.
[113] GREGORY L A, THIELENS N M, ARLAUD G J, et al. X-ray structure of the Ca2+-binding interaction domain of C1s. Insights into the assembly of the C1 complex of complement[J]. J Biol Chem, 2003, 278(34): 32157-32164.
[114] BALLY I, ROSSI V, LUNARDI T, et al. Identification of the C1q-binding sites of human C1r and C1s: a refined three-dimensional model of the C1 complex of complement[J]. J Biol Chem, 2009, 284(29): 19340-19348.
[115] SAHOO B K, LIN Y C, TU C F, et al. Signal peptide-CUB-EGF-like repeat-containing protein 1-promoted FLT3 signaling is critical for the initiation and maintenance of MLL-rearranged acute leukemia[J]. Haematologica, 2023, 108(5): 1284-1299.
[116] LIN Y C, ROFFLER S R, YAN Y T, et al. Disruption of Scube2 impairs endochondral bone formation[J]. J Bone Miner Res, 2015, 30(7): 1255-1267.
[117] LIN Y C, CHEN C C, CHENG C J, et al. Domain and functional analysis of a novel breast tumor suppressor protein, SCUBE2[J]. J Biol Chem, 2011, 286(30): 27039-27047.
[118] LIAO W J, LIN H, CHENG C F, et al. SCUBE1-enhanced bone morphogenetic protein signaling protects against renal ischemia-reperfusion injury[J]. Biochim Biophys Acta Mol Basis Dis, 2019, 1865(2): 329-338.
[119] LIN Y C, LEE Y C, LI L H, et al. Tumor suppressor SCUBE2 inhibits breast-cancer cell migration and invasion through the reversal of epithelial-mesenchymal transition[J]. J Cell Sci, 2014, 127(Pt 1): 85-100.
[120] LIN X. Functions of heparan sulfate proteoglycans in cell signaling during development[J]. Development, 2004, 131(24): 6009-6021.
[121] BELLAICHE Y, THE I, PERRIMON N. Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion[J]. Nature, 1998, 394(6688): 85-88.
[122] TAKEI Y, OZAWA Y, SATO M, et al. Three Drosophila EXT genes shape morphogen gradients through synthesis of heparan sulfate proteoglycans[J]. Development, 2004, 131(1): 73-82.
[123] HAN C, BELENKAYA T Y, WANG B, et al. Drosophila glypicans control the cell-to-cell movement of Hedgehog by a dynamin-independent process[J]. Development, 2004, 131(3): 601-611.
[124] LIN X, WEI G, SHI Z, et al. Disruption of gastrulation and heparan sulfate biosynthesis in EXT1-deficient mice[J]. Dev Biol, 2000, 224(2): 299-311.
[125] RUBIN J B, CHOI Y, SEGAL R A. Cerebellar proteoglycans regulate sonic hedgehog responses during development[J]. Development, 2002, 129(9): 2223-2232.
[126] WHALEN D M, MALINAUSKAS T, GILBERT R J, et al. Structural insights into proteoglycan-shaped Hedgehog signaling[J]. Proc Natl Acad Sci U S A, 2013, 110(41): 16420-16425.
[127] CHUANG P T, MCMAHON A P. Vertebrate Hedgehog signalling modulated by induction of a Hedgehog-binding protein[J]. Nature, 1999, 397(6720): 617-621.
[128] KWONG L, BIJLSMA M F, ROELINK H. Shh-mediated degradation of Hhip allows cell autonomous and non-cell autonomous Shh signalling[J]. Nat Commun, 2014, 5: 4849.
[129] AGRAWAL V, KIM D Y, KWON Y G. Hhip regulates tumor-stroma-mediated upregulation of tumor angiogenesis[J]. Exp Mol Med, 2017, 49(1): e289.
[130] GRIFFITHS S C, SCHWAB R A, EL OMARI K, et al. Hedgehog-interacting protein is a multimodal antagonist of Hedgehog signalling[J]. Nat Commun, 2021, 12(1): 7171.
[131] HOLTZ A M, GRIFFITHS S C, DAVIS S J, et al. Secreted HHIP1 interacts with heparan sulfate and regulates Hedgehog ligand localization and function[J]. J Cell Biol, 2015, 209(5): 739-757.
[132] BISHOP B, ARICESCU A R, HARLOS K, et al. Structural insights into hedgehog ligand sequestration by the human hedgehog-interacting protein HHIP[J]. Nat Struct Mol Biol, 2009, 16(7): 698-703.
[133] JAKOBS P, SCHULZ P, ORTMANN C, et al. Bridging the gap: heparan sulfate and Scube2 assemble Sonic hedgehog release complexes at the surface of producing cells[J]. Sci Rep, 2016, 6: 26435.
[134] JAKOBS P, SCHULZ P, SCHURMANN S, et al. Ca(2+) coordination controls sonic hedgehog structure and its Scube2-regulated release[J]. J Cell Sci, 2017, 130(19): 3261-3271.
[135] OHLIG S, FARSHI P, PICKHINKE U, et al. Sonic hedgehog shedding results in functional activation of the solubilized protein[J]. Dev Cell, 2011, 20(6): 764-774.
[136] TENZEN T, ALLEN B L, COLE F, et al. The cell surface membrane proteins Cdo and Boc are components and targets of the Hedgehog signaling pathway and feedback network in mice[J]. Dev Cell, 2006, 10(5): 647-656.
[137] ALLEN B L, TENZEN T, MCMAHON A P. The Hedgehog-binding proteins Gas1 and Cdo cooperate to positively regulate Shh signaling during mouse development[J]. Genes Dev, 2007, 21(10): 1244-1257.
[138] YAO S, LUM L, BEACHY P. The ihog cell-surface proteins bind Hedgehog and mediate pathway activation[J]. Cell, 2006, 125(2): 343-357.
[139] MCLELLAN J S, YAO S, ZHENG X, et al. Structure of a heparin-dependent complex of Hedgehog and Ihog[J]. Proc Natl Acad Sci U S A, 2006, 103(46): 17208-17213.
[140] MCLELLAN J S, ZHENG X, HAUK G, et al. The mode of Hedgehog binding to Ihog homologues is not conserved across different phyla[J]. Nature, 2008, 455(7215): 979-983.
[141] ZHANG Y, BEACHY P A. Cellular and molecular mechanisms of Hedgehog signalling[J]. Nat Rev Mol Cell Biol, 2023.
[142] ZHENG X, MANN R K, SEVER N, et al. Genetic and biochemical definition of the Hedgehog receptor[J]. Genes Dev, 2010, 24(1): 57-71.
[143] IZZI L, LEVESQUE M, MORIN S, et al. Boc and Gas1 each form distinct Shh receptor complexes with Ptch1 and are required for Shh-mediated cell proliferation[J]. Dev Cell, 2011, 20(6): 788-801.
[144] ROSTI K, GOLDMAN A, KAJANDER T. Solution structure and biophysical characterization of the multifaceted signalling effector protein growth arrest specific-1[J]. BMC Biochem, 2015, 16: 8.
[145] HUANG P, WIERBOWSKI B M, LIAN T, et al. Structural basis for catalyzed assembly of the Sonic hedgehog-Patched1 signaling complex[J]. Dev Cell, 2022.
[146] QI X, SCHMIEGE P, COUTAVAS E, et al. Two Patched molecules engage distinct sites on Hedgehog yielding a signaling-competent complex[J]. Science, 2018, 362(6410).
[147] INGHAM P W. Hedgehog signaling[M]. Cell signaling pathways in development. 2022: 1-58.
[148] STONE D M, HYNES M, ARMANINI M, et al. The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog[J]. Nature, 1996, 384(6605): 129-134.
[149] DENEF N, NEUBUSER D, PEREZ L, et al. Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened[J]. Cell, 2000, 102(4): 521-531.
[150] CORBIT K C, AANSTAD P, SINGLA V, et al. Vertebrate Smoothened functions at the primary cilium[J]. Nature, 2005, 437(7061): 1018-1021.
[151] HUANGFU D, ANDERSON K V. Cilia and Hedgehog responsiveness in the mouse[J]. Proc Natl Acad Sci U S A, 2005, 102(32): 11325-11330.
[152] ROHATGI R, MILENKOVIC L, SCOTT M P. Patched1 regulates hedgehog signaling at the primary cilium[J]. Science, 2007, 317(5836): 372-376.
[153] LI X Z, NIKAIDO H. Efflux-mediated drug resistance in bacteria: an update[J]. Drugs, 2009, 69(12): 1555-1623.
[154] SU C C, LI M, GU R, et al. Conformation of the AcrB multidrug efflux pump in mutants of the putative proton relay pathway[J]. J Bacteriol, 2006, 188(20): 7290-7296.
[155] LOPEZ M E, SCOTT M P. Genetic dissection of a cell-autonomous neurodegenerative disorder: lessons learned from mouse models of Niemann-Pick disease type C[J]. Dis Model Mech, 2013, 6(5): 1089-1100.
[156] MICHAUX G, GANSMULLER A, HINDELANG C, et al. CHE-14, a protein with a sterol-sensing domain, is required for apical sorting in C. elegans ectodermal epithelial cells[J]. Curr Biol, 2000, 10(18): 1098-1107.
[157] CHEN J K, TAIPALE J, COOPER M K, et al. Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened[J]. Genes Dev, 2002, 16(21): 2743-2748.
[158] CORCORAN R B, SCOTT M P. Oxysterols stimulate Sonic hedgehog signal transduction and proliferation of medulloblastoma cells[J]. Proc Natl Acad Sci U S A, 2006, 103(22): 8408-8413.
[159] MYERS B R, SEVER N, CHONG Y C, et al. Hedgehog pathway modulation by multiple lipid binding sites on the smoothened effector of signal response[J]. Dev Cell, 2013, 26(4): 346-357.
[160] HUANG P, NEDELCU D, WATANABE M, et al. Cellular cholesterol directly activates Smoothened in Hedgehog signaling[J]. Cell, 2016, 166(5): 1176-1187 e1114.
[161] GONG X, QIAN H, CAO P, et al. Structural basis for the recognition of Sonic Hedgehog by human Patched1[J]. Science, 2018, 361(6402).
[162] KUMAR N, SU C C, CHOU T H, et al. Crystal structures of the burkholderia multivorans hopanoid transporter HpnN[J]. Proc Natl Acad Sci U S A, 2017, 114(25): 6557-6562.
[163] ZHANG Y, BULKLEY D P, XIN Y, et al. Structural basis for cholesterol transport-like activity of the Hedgehog receptor Patched[J]. Cell, 2018, 175(5): 1352-1364 e1314.
[164] TUKACHINSKY H, PETROV K, WATANABE M, et al. Mechanism of inhibition of the tumor suppressor Patched by Sonic Hedgehog[J]. Proc Natl Acad Sci U S A, 2016, 113(40): E5866-E5875.
[165] KONITSIOTIS A D, CHANG S C, JOVANOVIC B, et al. Attenuation of hedgehog acyltransferase-catalyzed sonic Hedgehog palmitoylation causes reduced signaling, proliferation and invasiveness of human carcinoma cells[J]. PLoS One, 2014, 9(3): e89899.
[166] PETROVA E, RIOS-ESTEVES J, OUERFELLI O, et al. Inhibitors of Hedgehog acyltransferase block Sonic Hedgehog signaling[J]. Nat Chem Biol, 2013, 9(4): 247-249.
[167] GOETZ J A, SINGH S, SUBER L M, et al. A highly conserved amino-terminal region of sonic hedgehog is required for the formation of its freely diffusible multimeric form[J]. J Biol Chem, 2006, 281(7): 4087-4093.
[168] QIAN H, CAO P, HU M, et al. Inhibition of tetrameric Patched1 by Sonic Hedgehog through an asymmetric paradigm[J]. Nat Commun, 2019, 10(1): 2320.
[169] ZHANG Y, LU W J, BULKLEY D P, et al. Hedgehog pathway activation through nanobody-mediated conformational blockade of the Patched sterol conduit[J]. Proc Natl Acad Sci U S A, 2020, 117(46): 28838-28846.
[170] KINNEBREW M, IVERSON E J, PATEL B B, et al. Cholesterol accessibility at the ciliary membrane controls hedgehog signaling[J]. Elife, 2019, 8.
[171] LIU S L, SHENG R, JUNG J H, et al. Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol[J]. Nat Chem Biol, 2017, 13(3): 268-274.
[172] DESHPANDE I, LIANG J, HEDEEN D, et al. Smoothened stimulation by membrane sterols drives Hedgehog pathway activity[J]. Nature, 2019, 571(7764): 284-288.
[173] KINNEBREW M, LUCHETTI G, SIRCAR R, et al. Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes[J]. Elife, 2021, 10.
[174] MYERS B R, NEAHRING L, ZHANG Y, et al. Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium[J]. Proc Natl Acad Sci U S A, 2017, 114(52): E11141-E11150.
[175] RADHAKRISHNAN A, ROHATGI R, SIEBOLD C. Cholesterol access in cellular membranes controls Hedgehog signaling[J]. Nat Chem Biol, 2020, 16(12): 1303-1313.
[176] BYRNE E F X, SIRCAR R, MILLER P S, et al. Structural basis of Smoothened regulation by its extracellular domains[J]. Nature, 2016, 535(7613): 517-522.
[177] NEDELCU D, LIU J, XU Y, et al. Oxysterol binding to the extracellular domain of Smoothened in Hedgehog signaling[J]. Nat Chem Biol, 2013, 9(9): 557-564.
[178] LUCHETTI G, SIRCAR R, KONG J H, et al. Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling[J]. Elife, 2016, 5.
[179] XIAO X, TANG J J, PENG C, et al. Cholesterol modification of Smoothened is required for Hedgehog signaling[J]. Mol Cell, 2017, 66(1): 154-162 e110.
[180] HU A, ZHANG J Z, WANG J, et al. Cholesterylation of Smoothened is a calcium-accelerated autoreaction involving an intramolecular ester intermediate[J]. Cell Res, 2022, 32(3): 288-301.
[181] HUANG P, ZHENG S, WIERBOWSKI B M, et al. Structural basis of Smoothened activation in Hedgehog signaling[J]. Cell, 2018, 174(2): 312-324 e316.
[182] QI X, LIU H, THOMPSON B, et al. Cryo-EM structure of oxysterol-bound human Smoothened coupled to a heterotrimeric G(i)[J]. Nature, 2019, 571(7764): 279-283.
[183] YAUCH R L, DIJKGRAAF G J, ALICKE B, et al. Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma[J]. Science, 2009, 326(5952): 572-574.
[184] NACHTERGAELE S, WHALEN D M, MYDOCK L K, et al. Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling[J]. Elife, 2013, 2: e01340.
[185] DUNAEVA M, MICHELSON P, KOGERMAN P, et al. Characterization of the physical interaction of Gli proteins with SUFU proteins[J]. J Biol Chem, 2003, 278(7): 5116-5122.
[186] SVARD J, HEBY-HENRICSON K, PERSSON-LEK M, et al. Genetic elimination of Suppressor of fused reveals an essential repressor function in the mammalian Hedgehog signaling pathway[J]. Dev Cell, 2006, 10(2): 187-197.
[187] JIA J, TONG C, WANG B, et al. Hedgehog signalling activity of Smoothened requires phosphorylation by protein kinase A and casein kinase I[J]. Nature, 2004, 432(7020): 1045-1050.
[188] LI S, LI S, HAN Y, et al. Regulation of Smoothened phosphorylation and high-level Hedgehog signaling activity by a plasma membrane associated kinase[J]. PLoS Biol, 2016, 14(6): e1002481.
[189] CHEN Y, SASAI N, MA G, et al. Sonic Hedgehog dependent phosphorylation by CK1alpha and GRK2 is required for ciliary accumulation and activation of smoothened[J]. PLoS Biol, 2011, 9(6): e1001083.
[190] LI S, MA G, WANG B, et al. Hedgehog induces formation of PKA-Smoothened complexes to promote Smoothened phosphorylation and pathway activation[J]. Sci Signal, 2014, 7(332): ra62.
[191] HAN Y, WANG B, CHO Y S, et al. Phosphorylation of Ci/Gli by fused family kinases promotes Hedgehog signaling[J]. Dev Cell, 2019, 50(5): 610-626 e614.
[192] LI S, CHEN Y, SHI Q, et al. Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila[J]. PLoS Biol, 2012, 10(1): e1001239.
[193] LI S, LI S, WANG B, et al. Hedgehog reciprocally controls trafficking of Smo and Ptc through the Smurf family of E3 ubiquitin ligases[J]. Sci Signal, 2018, 11(516).
[194] SGRO G G, COSTA T R D. Cryo-EM grid preparation of membrane protein samples for single particle analysis[J]. Front Mol Biosci, 2018, 5: 74.
[195] QI C, DI MININ G, VERCELLINO I, et al. Structural basis of sterol recognition by human hedgehog receptor PTCH1[J]. Sci Adv, 2019, 5(9): eaaw6490.
[196] FAN X, WANG J, ZHANG X, et al. Single particle cryo-EM reconstruction of 52 kDa streptavidin at 3.2 Angstrom resolution[J]. Nat Commun, 2019, 10(1): 2386.
[197] WU X, RAPOPORT T A. Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies)[J]. Proc Natl Acad Sci U S A, 2021, 118(41).