RESUMO
Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases (VEGFRs). The extracellular domain of VEGFRs consists of seven Ig-homology domains, of which D2-3 form the ligand-binding site, while the membrane proximal domains D4-7 are involved in homotypic interactions in ligand-bound receptor dimers. Based on low-resolution structures, we identified allosteric sites in D4-5 and D7 of vascular endothelial growth factor receptor 2 (VEGFR-2) accomplishing regulatory functions. Allosteric inhibition of VEGFR-2 signaling represents an attractive option for the treatment of neovascular diseases. We showed earlier that DARPin® binders to domains D4 or D7 are potent VEGFR-2 inhibitors. Here we investigated in detail the allosteric inhibition mechanism of the domain D4 binding inhibitor D4b. The 2.38 Å crystal structure of D4b in complex with VEGFR-2 D4-5, the first high-resolution structure of this VEGFR-2 segment, indicates steric hindrance by D4b as the mechanism of inhibition of receptor activation. At the cellular level, D4b triggered quantitative internalization of VEGFR-2 in the absence of ligand and thus clearance of VEGFR-2 from the surface of endothelial cells. The allosteric VEGFR-2 inhibition was sufficiently strong to efficiently inhibit the growth of human endothelial cells at suboptimal dose in a mouse xenograft model in vivo, underlining the therapeutic potential of the approach.
Assuntos
Inibidores da Angiogênese , Sistemas de Liberação de Medicamentos , Células Endoteliais da Veia Umbilical Humana , Neovascularização Patológica , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Cristalografia por Raios X , Células HEK293 , Xenoenxertos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Células Endoteliais da Veia Umbilical Humana/transplante , Humanos , Camundongos , Camundongos SCID , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Suínos , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five- to ten-fold. Expression of a SAS-6 mutant that forms six-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with eight- or nine-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel-microtubule interactions, this SAS-6 mutant produced six- to eight-fold symmetric cartwheels. Concurrently, the microtubule wall maintained eight- and nine-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in nine-fold symmetric centrioles that exhibited impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.
Assuntos
Proteínas de Algas/metabolismo , Centríolos/metabolismo , Chlamydomonas reinhardtii/metabolismo , Microtúbulos/metabolismo , Proteínas de Algas/química , Proteínas de Algas/genética , Western Blotting , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Centríolos/química , Centríolos/ultraestrutura , Chlamydomonas reinhardtii/genética , Cristalografia por Raios X , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia de Força Atômica , Microscopia Eletrônica , Microscopia de Fluorescência , Microtúbulos/química , Microtúbulos/ultraestrutura , Modelos Moleculares , Conformação Molecular , Mutação , Multimerização Proteica , Estrutura Terciária de Proteína , Interferência de RNARESUMO
Botulinum neurotoxin A causes botulism but is also used for medical and cosmetic applications. A detailed molecular understanding of BoNT/A--host receptor interactions is therefore fundamental for improving current clinical applications and for developing new medical strategies targeting human disorders. Towards this end, we recently solved an X-ray crystal structure of BoNT/A1 in complex with its neuronal protein receptor SV2C. Based on our findings, we discuss the potential implications for BoNT/A function.
