RESUMEN
Regulated protein degradation in eukaryotes is performed by the 26S proteasome, which contains a 19-subunit regulatory particle (RP) that binds, processes, and translocates substrates to a 28-subunit hollow core particle (CP) where proteolysis occurs. In addition to its intrinsic subunits, myriad proteins interact with the proteasome transiently, including factors that assist and/or regulate its degradative activities. Efforts to identify proteasome-interacting components and/or to solve its structure have relied on over-expression of a tagged plasmid, establishing stable cell lines, or laborious purification protocols to isolate native proteasomes from cells. Here, we describe an engineered human cell line, derived from colon cancer HCT116 cells, with a biotin handle on the RP subunit hRpn1/PSMD2 (proteasome 26S subunit, non-ATPase 2) for purification of 26S proteasomes. A 75-residue sequence from Propionibacterium shermanii that is biotinylated in mammalian cells was added following a tobacco etch virus protease cut site at the C terminus of hRpn1. We tested and found that 26S proteasomes can be isolated from this modified HCT116 cell line by using a simple purification protocol. More specifically, biotinylated proteasomes were purified from the cell lysates by using neutravidin agarose resin and released from the resin following incubation with tobacco etch virus protease. The purified proteasomes had equivalent activity in degrading a model ubiquitinated substrate, namely ubiquitinated p53, compared to commercially available bovine proteasomes that were purified by fractionation. In conclusion, advantages of this approach to obtain 26S proteasomes over others is the simple purification protocol and that all cellular proteins, including the tagged hRpn1 subunit, remain at endogenous stoichiometry.
Asunto(s)
Técnicas Citológicas , Complejo de la Endopetidasa Proteasomal , Animales , Bovinos , Humanos , Línea Celular , Citoplasma/metabolismo , Mamíferos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitina/metabolismo , Técnicas Citológicas/métodosRESUMEN
This case concerns an 18-month-old with masked congestive heart failure (CHF) from an unrepaired vein of Galen malformation and superior sinus venosus defect who progressed to severe, refractory CHF following superior sinus venosus defect repair. Partial transvenous coil embolization of a very-high-risk vein of Galen malformation resolved CHF symptoms. (Level of Difficulty: Advanced.).