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1.
J Cell Biol ; 177(1): 51-61, 2007 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-17420289

RESUMO

The ubiquitin (Ub) domain protein Herp plays a crucial role in the maintenance of calcium homeostasis during endoplasmic reticulum (ER) stress. We now show that Herp is a substrate as well as an activator of the E3 Ub ligase POSH. Herp-mediated POSH activation requires the Ubl domain and exclusively promotes lysine-63-linked polyubiquitination. Confocal microscopy demonstrates that Herp resides mostly in the trans-Golgi network, but, shortly after calcium perturbation by thapsigargin (Tpg), it appears mainly in the ER. Substitution of all lysine residues within the Ubl domain abolishes lysine-63-linked polyubiquitination of Herp in vitro and calcium-induced Herp relocalization that is also abrogated by the overexpression of a dominant-negative POSHV14A. A correlation exists between the kinetics of Tpg-induced Herp relocalization and POSH-dependent polyubiquitination. Finally, the overexpression of POSH attenuates, whereas the inhibition of POSH by the expression of POSHV14A or by RNA interference enhances Tpg-induced calcium burst. Altogether, these results establish a critical role for POSH-mediated ubiquitination in the maintenance of calcium homeostasis through the spatial control of Herp.


Assuntos
Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Células HeLa , Homeostase , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Membrana/química , Modelos Biológicos , Estrutura Terciária de Proteína , Tapsigargina/farmacologia , Tunicamicina/farmacologia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Rede trans-Golgi/metabolismo
2.
Proc Natl Acad Sci U S A ; 102(5): 1478-83, 2005 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-15659549

RESUMO

HIV type 1 (HIV-1) was shown to assemble either at the plasma membrane or in the membrane of late endosomes. Now, we report an essential role for human ubiquitin ligase POSH (Plenty of SH3s; hPOSH), a trans-Golgi network-associated protein, in the targeting of HIV-1 to the plasma membrane. Small inhibitory RNA-mediated silencing of hPOSH ablates virus secretion and Gag plasma membrane localization. Reintroduction of native, but not a RING finger mutant, hPOSH restores virus release and Gag plasma membrane localization in hPOSH-depleted cells. Furthermore, expression of the RING finger mutant hPOSH inhibits virus release and induces accumulation of intracellular Gag in normal cells. Together, our results identify a previously undescribed step in HIV biogenesis and suggest a direct function for hPOSH-mediated ubiquitination in protein sorting at the trans-Golgi network. Consequently, hPOSH may be a useful host target for therapeutic intervention.


Assuntos
HIV-1/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Replicação Viral/fisiologia , Rede trans-Golgi/enzimologia , Membrana Celular/enzimologia , Membrana Celular/virologia , Clonagem Molecular , Produtos do Gene gag/metabolismo , Inativação Gênica , Células HeLa , Humanos , Transporte Proteico , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína Ligases/genética
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