RESUMO
The nuclear pore complex (NPC) controls the transport of macromolecules between the nucleus and cytoplasm, but its molecular architecture has thus far remained poorly defined. We biochemically reconstituted NPC core protomers and elucidated the underlying protein-protein interaction network. Flexible linker sequences, rather than interactions between the structured core scaffold nucleoporins, mediate the assembly of the inner ring complex and its attachment to the NPC coat. X-ray crystallographic analysis of these scaffold nucleoporins revealed the molecular details of their interactions with the flexible linker sequences and enabled construction of full-length atomic structures. By docking these structures into the cryoelectron tomographic reconstruction of the intact human NPC and validating their placement with our nucleoporin interactome, we built a composite structure of the NPC symmetric core that contains ~320,000 residues and accounts for ~56 megadaltons of the NPC's structured mass. Our approach provides a paradigm for the structure determination of similarly complex macromolecular assemblies.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Mapas de Interação de Proteínas , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Microscopia Crioeletrônica , Cristalografia por Raios X , Citoplasma/metabolismo , Tomografia com Microscopia Eletrônica , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Dados de Sequência Molecular , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. We present the reconstitution and interdisciplinary analyses of the ~425-kilodalton inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1â¢Nup49â¢Nup57 channel nucleoporin heterotrimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96. The CNTâ¢Nic96 structure reveals that Nic96 functions as an assembly sensor that recognizes the three-dimensional architecture of the CNT, thereby mediating the incorporation of a defined CNT state into the NPC. We propose that the IRC adopts a relatively rigid scaffold that recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel dilation.
Assuntos
Chaetomium/ultraestrutura , Proteínas Fúngicas/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Poro Nuclear/ultraestrutura , Proteínas Nucleares/ultraestrutura , Sequência de Aminoácidos , Chaetomium/metabolismo , Proteínas Fúngicas/química , Dados de Sequência Molecular , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas Nucleares/química , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: The BIO14.6 hamster provides a useful model of hereditary cardiomyopathies and muscular dystrophy. Previous δ-sarcoglycan (δSG) gene therapy (GT) studies were limited to neonatal and young adult animals and prevented the development of cardiac and skeletal muscle dysfunction. GT of a pseudophosphorylated mutant of phospholamban (S16EPLN) moderately alleviated the progression of cardiomyopathy. METHODS AND RESULTS: We treated 4-month-old BIO14.6 hamsters with established cardiac and skeletal muscle diseases intravenously with a serotype-9 adeno-associated viral vector carrying δSG alone or in combination with S16EPLN. Before treatment at age 14 weeks, the left ventricular fractional shortening by echocardiography was 31.3% versus 45.8% in normal hamsters. In a randomized trial, GT halted progression of left ventricular dilation and left ventricular dysfunction. Also, respiratory function improved. Addition of S16EPLN had no significant additional effects. δSG-GT prevented severe degeneration of the transverse tubular system in cardiomyocytes (electron tomography) and restored distribution of dystrophin and caveolin-3. All placebo-treated hamsters, except animals removed for the hemodynamic study, died with heart failure between 34 and 67 weeks of age. In the GT group, signs of cardiac and respiratory failure did not develop, and animals lived for 92 weeks or longer, an age comparable to that reported in normal hamsters. CONCLUSION: GT was highly effective in BIO14.6 hamsters even when given in late-stage disease, a finding that may carry implications for the future treatment of hereditary cardiac and muscle diseases in humans.