The nuclear replication and retention functions of the Epstein-Barr virus (EBV) have been utilized here to maintain retroviral constructs episomally within human cell-based retroviral packaging lines. These hybrid EBV/retroviral constructs are capable of producing helper-free recombinant retrovirus as soon as 48 hr and for at least 30 days after transfection into 293T-based ecotropic and/or amphotropic retroviral packaging cells. Viral titers greater than 10(7) TU/ml were obtained after puromycin selection of transfected retroviral packaging cells. This episomal approach to retroviral production circumvents some limitations inherent in transient and chromosomally stable retroviral producer systems, affording reproducibly rapid, large-scale, stable, and high-titer retrovirus production.

Ligand-induced protein degradation has emerged as a compelling approach to promote the targeted elimination of proteins from cells by directing these proteins to the ubiquitin-proteasome machinery. So far, only a limited number of E3 ligases have been found to support ligand-induced protein degradation, reflecting a dearth of E3-binding compounds for proteolysis-targeting chimera (PROTAC) design. Here, we describe a functional screening strategy performed with a focused library of candidate electrophilic PROTACs to discover bifunctional compounds that degrade proteins in human cells by covalently engaging E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs act through modifying specific cysteines in DCAF11, a poorly characterized E3 ligase substrate adaptor. We further show that DCAF11-directed electrophilic PROTACs can degrade multiple endogenous proteins, including FBKP12 and the androgen receptor, in human prostate cancer cells. Our findings designate DCAF11 as an E3 ligase capable of supporting ligand-induced protein degradation via electrophilic PROTACs.

Modulation of mitochondrial function through inhibiting respiratory complex I activates a key sensor of cellular energy status, the 5'-AMP-activated protein kinase (AMPK). Activation of AMPK results in the mobilization of nutrient uptake and catabolism for mitochondrial ATP generation to restore energy homeostasis. How these nutrient pathways are affected in the presence of a potent modulator of mitochondrial function and the role of AMPK activation in these effects remain unclear. We have identified a molecule, named R419, that activates AMPK in vitro via complex I inhibition at much lower concentrations than metformin (IC50 100 nM vs 27 mM, respectively). R419 potently increased myocyte glucose uptake that was dependent on AMPK activation, while its ability to suppress hepatic glucose production in vitro was not. In addition, R419 treatment of mouse primary hepatocytes increased fatty acid oxidation and inhibited lipogenesis in an AMPK-dependent fashion. We have performed an extensive metabolic characterization of its effects in the db/db mouse diabetes model. In vivo metabolite profiling of R419-treated db/db mice showed a clear upregulation of fatty acid oxidation and catabolism of branched chain amino acids. Additionally, analyses performed using both 13C-palmitate and 13C-glucose tracers revealed that R419 induces complete oxidation of both glucose and palmitate to CO2 in skeletal muscle, liver, and adipose tissue, confirming that the compound increases mitochondrial function in vivo. Taken together, our results show that R419 is a potent inhibitor of complex I and modulates mitochondrial function in vitro and in diabetic animals in vivo. R419 may serve as a valuable molecular tool for investigating the impact of modulating mitochondrial function on nutrient metabolism in multiple tissues and on glucose and lipid homeostasis in diabetic animal models.

INTRODUCTION Mechanical ventilation (MV) is an important component of modern medical practice which allows support of breathing in the intensive care unit (ICU) and during surgery requiring general anesthesia. Many patients, however, fail initial weaning from the ventilator and enter the difficult realm of prolonged ventilation. Patients who develop this ventilator dependence, though a diverse group, share the common underlying problem of substantial dysfunction of the major inspiratory muscle, the diaphragm (1-8). The development of ventilator-induced diaphragm dysfunction (VIDD) appears to be a major underlying cause of prolonged ventilator-dependence with its attendant dramatic increase in morbidity and mortality (9-14). The pathogenesis of VIDD includes both atrophy of diaphragmatic myofibers and loss of diaphragmatic contractile function (that is, specific force) unrelated to atrophy (15-17). In previous studies, MV with diaphragm inactivity has been shown to elicit significant dysfunction and/or atrophy of myofibers in the diaphragm of humans (18-21), rats (22), mice (23-25), rabbits (26) and piglets (27). With regard to the atrophy, several proteolytic events, such as activation of the ubiquitin proteasome system (UPS) (28-30), autophagy (24,25,31) and apoptosis (32-35), and upregulation of calpain (36), have been demonstrated in MV models. We and others have re