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Nat Commun ; 11(1): 1621, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32238803


Activin receptor-like kinase 1 (ALK1)-mediated endothelial cell signalling in response to bone morphogenetic protein 9 (BMP9) and BMP10 is of significant importance in cardiovascular disease and cancer. However, detailed molecular mechanisms of ALK1-mediated signalling remain unclear. Here, we report crystal structures of the BMP10:ALK1 complex at 2.3 Å and the prodomain-bound BMP9:ALK1 complex at 3.3 Å. Structural analyses reveal a tripartite recognition mechanism that defines BMP9 and BMP10 specificity for ALK1, and predict that crossveinless 2 is not an inhibitor of BMP9, which is confirmed by experimental evidence. Introduction of BMP10-specific residues into BMP9 yields BMP10-like ligands with diminished signalling activity in C2C12 cells, validating the tripartite mechanism. The loss of osteogenic signalling in C2C12 does not translate into non-osteogenic activity in vivo and BMP10 also induces bone-formation. Collectively, these data provide insight into ALK1-mediated BMP9 and BMP10 signalling, facilitating therapeutic targeting of this important pathway.

Receptores de Activinas Tipo II/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Transdução de Sinais/fisiologia , Receptores de Activinas Tipo II/química , Animais , Sítios de Ligação , Proteínas Morfogenéticas Ósseas/química , Osso e Ossos/química , Osso e Ossos/metabolismo , Linhagem Celular , Cristalografia por Raios X , Células Endoteliais/metabolismo , Fator 2 de Diferenciação de Crescimento/química , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Fator de Crescimento Transformador beta/metabolismo
Proc Natl Acad Sci U S A ; 116(36): 17800-17808, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31431534


Endoglin (ENG) is a coreceptor of the transforming growth factor-ß (TGFß) family signaling complex, which is highly expressed on endothelial cells and plays a key role in angiogenesis. Its extracellular domain can be cleaved and released into the circulation as soluble ENG (sENG). High circulating levels of sENG contribute to the pathogenesis of preeclampsia (PE). Circulating bone morphogenetic protein 9 (BMP9), a vascular quiescence and endothelial-protective factor, binds sENG with high affinity, but how sENG participates in BMP9 signaling complexes is not fully resolved. sENG was thought to be a ligand trap for BMP9, preventing type II receptor binding and BMP9 signaling. Here we show that, despite cell-surface ENG being a dimer linked by disulfide bonds, sENG purified from human placenta and plasma from PE patients is primarily in a monomeric form. Incubating monomeric sENG with the circulating form of BMP9 (prodomain-bound form) in solution leads to the release of the prodomain and formation of a sENG:BMP9 complex. Furthermore, we demonstrate that binding of sENG to BMP9 does not inhibit BMP9 signaling. Indeed, the sENG:BMP9 complex signals with comparable potency and specificity to BMP9 on human primary endothelial cells. The full signaling activity of the sENG:BMP9 complex required transmembrane ENG. This study confirms that rather than being an inhibitory ligand trap, increased circulating sENG might preferentially direct BMP9 signaling via cell-surface ENG at the endothelium. This is important for understanding the role of sENG in the pathobiology of PE and other cardiovascular diseases.

Endoglina/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Proteínas da Gravidez/metabolismo , Transdução de Sinais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Humanos , Placenta/patologia , Pré-Eclâmpsia/patologia , Gravidez
Cell Rep ; 25(10): 2866-2877.e5, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30517872


Tubulin glutamylation is a reversible posttranslational modification that accumulates on stable microtubules (MTs). While abnormally high levels of this modification lead to a number of disorders such as male sterility, retinal degeneration, and neurodegeneration, very little is known about the molecular mechanisms underlying the regulation of glutamylase activity. Here, we found that CSAP forms a complex with TTLL5, and we demonstrate that the two proteins regulate their reciprocal abundance. Moreover, we show that CSAP increases TTLL5-mediated glutamylation and identify the TTLL5-interacting domain. Deletion of this domain leads to complete loss of CSAP activating function without impacting its MT binding. Binding of CSAP to TTLL5 promotes relocalization of TTLL5 toward MTs. Finally, we show that CSAP binds and activates all of the remaining autonomously active TTLL glutamylases. As such, we present CSAP as a major regulator of tubulin glutamylation and associated functions.

Proteínas de Transporte/metabolismo , Glutamatos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Linhagem Celular , Humanos , Ligação Proteica , Tubulina (Proteína)/metabolismo
J Biol Chem ; 291(6): 2954-66, 2016 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-26631724


BMP10 is highly expressed in the developing heart and plays essential roles in cardiogenesis. BMP10 deletion in mice results in embryonic lethality because of impaired cardiac development. In adults, BMP10 expression is restricted to the right atrium, though ventricular hypertrophy is accompanied by increased BMP10 expression in a rat hypertension model. However, reports of BMP10 activity in the circulation are inconclusive. In particular, it is not known whether in vivo secreted BMP10 is active or whether additional factors are required to achieve its bioactivity. It has been shown that high-affinity binding of the BMP10 prodomain to the mature ligand inhibits BMP10 signaling activity in C2C12 cells, and it was proposed that prodomain-bound BMP10 (pBMP10) complex is latent. In this study, we demonstrated that the BMP10 prodomain did not inhibit BMP10 signaling activity in multiple endothelial cells, and that recombinant human pBMP10 complex, expressed in mammalian cells and purified under native conditions, was fully active. In addition, both BMP10 in human plasma and BMP10 secreted from the mouse right atrium were fully active. Finally, we confirmed that active BMP10 secreted from mouse right atrium was in the prodomain-bound form. Our data suggest that circulating BMP10 in adults is fully active and that the reported vascular quiescence function of BMP10 in vivo is due to the direct activity of pBMP10 and does not require an additional activation step. Moreover, being an active ligand, recombinant pBMP10 may have therapeutic potential as an endothelial-selective BMP ligand, in conditions characterized by loss of BMP9/10 signaling.

Proteínas Morfogenéticas Ósseas/metabolismo , Cardiomegalia/metabolismo , Células Endoteliais/metabolismo , Transdução de Sinais , Animais , Proteínas Morfogenéticas Ósseas/genética , Cardiomegalia/genética , Cardiomegalia/patologia , Linhagem Celular , Células Endoteliais/patologia , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Humanos , Camundongos , Ratos
Cell ; 138(2): 366-76, 2009 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-19632184


Microtubules are filamentous polymers essential for cell viability. Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, migration, and morphogenesis. EB1 and its homologs are highly conserved proteins that play an important role in the targeting of +TIPs to microtubule ends, but the underlying molecular mechanism remains elusive. By using live cell experiments and in vitro reconstitution assays, we demonstrate that a short polypeptide motif, Ser-x-Ile-Pro (SxIP), is used by numerous +TIPs, including the tumor suppressor APC, the transmembrane protein STIM1, and the kinesin MCAK, for localization to microtubule tips in an EB1-dependent manner. Structural and biochemical data reveal the molecular basis of the EB1-SxIP interaction and explain its negative regulation by phosphorylation. Our findings establish a general "microtubule tip localization signal" (MtLS) and delineate a unifying mechanism for this subcellular protein targeting process.

Proteínas Associadas aos Microtúbulos/química , Microtúbulos/química , Sinais Direcionadores de Proteínas , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Alinhamento de Sequência