Fbxo6 confers drug-sensitization to cisplatin via inhibiting the activation of Chk1 in non-small cell lung cancer.

Fbxo6 (also called FBG2) is a critical component of the evolutionarily conserved ubiquitin-protein ligase complex SCF (Skp1/Cdc53-Cullin1/F-box). Previous studies have demonstrated that Fbxo6 facilitates the growth and proliferation but inhibits the apoptosis and invasion of gastric cancer cells. However, the role of Fbxo6 in non-small cell lung cancer (NSCLC) is still not clear. Our results revealed that Fbxo6 expression is correlated with early TNM stage and favorable overall survival of NSCLC patients. Further in vitro experiments showed that Fbxo6 inhibits proliferation, facilitates apoptosis and promotes the sensitivity of cisplatin via decreased expression and phosphorylation of Chk1. Thus, Fbxo6 may be a useful prognosis marker and therapeutic target to overcome the chemoresistance of cisplatin-based chemotherapy agents in NSCLC patients.

The subunit assembly state of the Mediator complex is nutrient-regulated and is dysregulated in a genetic model of insulin resistance and obesity.

The Mediator complex plays a critical role in the regulation of transcription by linking transcription factors to RNA polymerase II. By examining mouse livers, we have found that in the fasted state, the Mediator complex exists primarily as an approximately 1.2-MDa complex, consistent with the size of the large Mediator complex, whereas following feeding, it converts to an approximately 600-kDa complex, consistent with the size of the core Mediator complex. This dynamic change is due to the dissociation and degradation of the kinase module that includes the MED13, MED12, cyclin-dependent kinase 8 (CDK8), and cyclin C (CCNC) subunits. The dissociation and degradation of the kinase module are dependent upon nutrient activation of mTORC1 that is necessary for the induction of lipogenic gene expression because pharmacological or genetic inhibition of mTORC1 in the fed state restores the kinase module. The degradation but not dissociation of the kinase module depends upon the E3 ligase, SCFFBW7 In addition, genetically insulin-resistant and obese db/db mice in the fasted state displayed elevated lipogenic gene expression and loss of the kinase module that was reversed following mTORC1 inhibition. These data demonstrate that the assembly state of the Mediator complex undergoes physiologic regulation during normal cycles of fasting and feeding in the mouse liver. Furthermore, the assembly state of the Mediator complex is dysregulated in states of obesity and insulin resistance.

FBXO32 activates NF-κB through IκBα degradation in inflammatory and genotoxic stress.

In response to diverse stresses, the canonical NF-κB pathway gets activated primarily to protect the cells and maintain their genomic integrity. It activates the cell cycle checkpoints allowing the cells with limited damage to restore a normal life cycle. One of the key events in activation of the canonical NF-κB pathway is the selective proteasomal degradation of IκBα. It has been previously shown that F-box protein ßTRCP1 has limited role in directing the proteasomal degradation of IκBα during stress conditions. Here, we report another member of F-box family proteins, FBXO32, as a potential activator of NF-κB signaling during genotoxic stress and inflammatory response. Following genotoxic or inflammatory stress, FBXO32 is stabilized, which leads to polyubiquitination and proteasome mediated degradation of IκBα. We also found that FBXO32 is required for physiological regulation of IκBα levels in unstressed cells. Thus, we decipher the new role of FBXO32 in regulation of NF-κB signaling pathway.

Atrogin-1 deficiency promotes cardiomyopathy and premature death via impaired autophagy.

Cardiomyocyte proteostasis is mediated by the ubiquitin/proteasome system (UPS) and autophagy/lysosome system and is fundamental for cardiac adaptation to both physiologic (e.g., exercise) and pathologic (e.g., pressure overload) stresses. Both the UPS and autophagy/lysosome system exhibit reduced efficiency as a consequence of aging, and dysfunction in these systems is associated with cardiomyopathies. The muscle-specific ubiquitin ligase atrogin-1 targets signaling proteins involved in cardiac hypertrophy for degradation. Here, using atrogin-1 KO mice in combination with in vivo pulsed stable isotope labeling of amino acids in cell culture proteomics and biochemical and cellular analyses, we identified charged multivesicular body protein 2B (CHMP2B), which is part of an endosomal sorting complex (ESCRT) required for autophagy, as a target of atrogin-1-mediated degradation. Mice lacking atrogin-1 failed to degrade CHMP2B, resulting in autophagy impairment, intracellular protein aggregate accumulation, unfolded protein response activation, and subsequent cardiomyocyte apoptosis, all of which increased progressively with age. Cellular proteostasis alterations resulted in cardiomyopathy characterized by myocardial remodeling with interstitial fibrosis, with reduced diastolic function and arrhythmias. CHMP2B downregulation in atrogin-1 KO mice restored autophagy and decreased proteotoxicity, thereby preventing cell death. These data indicate that atrogin-1 promotes cardiomyocyte health through mediating the interplay between UPS and autophagy/lysosome system and its alteration promotes development of cardiomyopathies.

MAFbx/Atrogin-1 is required for atrophic remodeling of the unloaded heart.

BACKGROUND: Mechanical unloading of the failing human heart induces profound cardiac changes resulting in the reversal of a distorted structure and function. In this process, cardiomyocytes break down unneeded proteins and replace those with new ones. The specificity of protein degradation via the ubiquitin proteasome system is regulated by ubiquitin ligases. Over-expressing the ubiquitin ligase MAFbx/Atrogin-1 in the heart inhibits the development of cardiac hypertrophy, but the role of MAFbx/Atrogin-1 in the unloaded heart is not known. METHODS AND RESULTS: Mechanical unloading, by heterotopic transplantation, decreased heart weight and cardiomyocyte cross-sectional area in wild type mouse hearts. Unexpectedly, MAFbx/Atrogin-1(-/-) hearts hypertrophied after transplantation (n=8-10). Proteasome activity and markers of autophagy were increased to the same extent in WT and MAFbx/Atrogin-1(-/-) hearts after transplantation (unloading). Calcineurin, a regulator of cardiac hypertrophy, was only upregulated in MAFbx/Atrogin-1(-/-) transplanted hearts, while the mTOR pathway was similarly activated in unloaded WT and MAFbx/Atrogin-1(-/-) hearts. MAFbx/Atrogin-1(-/-) cardiomyocytes exhibited increased calcineurin protein expression, NFAT transcriptional activity, and protein synthesis rates, while inhibition of calcineurin normalized NFAT activity and protein synthesis. Lastly, mechanical unloading of failing human hearts with a left ventricular assist device (n=18) also increased MAFbx/Atrogin-1 protein levels and expression of NFAT regulated genes. CONCLUSIONS: MAFbx/Atrogin-1 is required for atrophic remodeling of the heart. During unloading, MAFbx/Atrogin-1 represses calcineurin-induced cardiac hypertrophy. Therefore, MAFbx/Atrogin-1 not only regulates protein degradation, but also reduces protein synthesis, exerting a dual role in regulating cardiac mass.

Deletion of atrophy enhancing genes fails to ameliorate the phenotype in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disease causing degeneration of lower motor neurons and muscle atrophy. One therapeutic avenue for SMA is targeting signaling pathways in muscle to ameliorate atrophy. Muscle Atrophy F-box, MAFbx, and Muscle RING Finger 1, MuRF1, are muscle-specific ubiquitin ligases upregulated in skeletal and cardiac muscle during atrophy. Homozygous knock-out of MAFbx or MuRF1 causes muscle sparing in adult mice subjected to atrophy by denervation. We wished to determine whether blockage of the major muscle atrophy pathways by deletion of MAFbx or MuRF1 in a mouse model of SMA would improve the phenotype. Deletion of MAFbx in the Δ7 SMA mouse model had no effect on the weight and the survival of the mice while deletion of MuRF1 was deleterious. MAFbx(-/-)-SMA mice showed a significant alteration in fiber size distribution tending towards larger fibers. In skeletal and cardiac tissue MAFbx and MuRF1 transcripts were upregulated whereas MuRF2 and MuRF3 levels were unchanged in Δ7 SMA mice. We conclude that deletion of the muscle ubiquitin ligases does not improve the phenotype of a Δ7 SMA mouse. Furthermore, it seems unlikely that the beneficial effect of HDAC inhibitors is mediated through inhibition of MAFbx and MuRF1.

Upregulation of proteasome activity in muscle RING finger 1-null mice following denervation.

Deletion of muscle RING finger 1 (MuRF1), an E3 ubiquitin ligase, leads to sparing of muscle mass following denervation. The purpose of this study was to test the hypothesis that muscle sparing in mice with a deletion of MuRF1 is due to the selective inhibition of the ubiquitin proteasome system. Activities of the 20S and 26S proteasomes, calpain and cathepsin L, were measured in the triceps surae muscles of wild-type (WT) and MuRF1-knockout (KO) mice at 3 and 14 d following denervation. In addition, fractional protein synthesis rates and differential gene expression were measured in WT and KO muscle. The major finding was that 20S and 26S proteasome activities were significantly elevated (1.5- to 2.5-fold) after 14 d of denervation in both WT and KO mice relative to control, but interestingly, the activities of both the 20S and 26S proteasome were significantly higher in KO than WT mice. Further, mRNA expression of MAFbx was elevated after 14 d of denervation in KO, but not WT, mice. These data challenge the conventional dogma that MuRF1 is controlling the degradation of only contractile proteins and suggest a role for MuRF1 in the global control of the ubiquitin proteasome system and protein turnover.

Endogenous muscle atrophy F-box mediates pressure overload-induced cardiac hypertrophy through regulation of nuclear factor-kappaB.

RATIONALE: Overexpression of muscle atrophy F-box (MAFbx/atrogin-1), an E3 ubiquitin ligase, induces proteasomal degradation in cardiomyocytes. The role of endogenous MAFbx in regulating cardiac hypertrophy and failure remains unclear. OBJECTIVE: We investigated the role of MAFbx in regulating cardiac hypertrophy and function in response to pressure overload. Transverse aortic constriction (TAC) was applied to MAFbx knockout (KO) and wild-type (WT) mice. METHODS AND RESULTS: Expression of MAFbx in WT mice was significantly increased by TAC. TAC-induced increases in cardiac hypertrophy were significantly smaller in MAFbx KO than in WT mice. There was significantly less lung congestion and interstitial fibrosis in MAFbx KO than in WT mice. MAFbx KO also inhibited ß-adrenergic cardiac hypertrophy. DNA microarray analysis revealed that activation of genes associated with the transcription factor binding site for the nuclear factor-κB family were inhibited in MAFbx KO mice compared with WT mice after TAC. Although the levels of IκB-α were significantly decreased after TAC in WT mice, they were increased in MAFbx KO mice. MAFbx regulates ubiquitination and proteasomal degradation of IκB-α in cardiomyocytes. In primary cultured rat cardiomyocytes, phenylephrine-induced activation of nuclear factor-κB and hypertrophy were significantly suppressed by MAFbx knockdown but were partially rescued by overexpression of nuclear factor-κB p65. CONCLUSIONS: MAFbx plays an essential role in mediating cardiac hypertrophy in response to pressure overload. Downregulation of MAFbx inhibits cardiac hypertrophy in part through stabilization of IκB-α and inactivation of nuclear factor-κB. Taken together, inhibition of MAFbx attenuates pathological hypertrophy, thereby protecting the heart from progression into heart failure.

Simian virus 40 large T antigen's association with the CUL7 SCF complex contributes to cellular transformation.

Simian virus 40 large T antigen (T Ag) is capable of immortalizing and transforming rodent cells. The transforming activity of T Ag is due in large part to perturbation of the tumor suppressor proteins p53 and the retinoblastoma (pRB) family members. Inactivation of these tumor suppressors may not be sufficient for T Ag-mediated cellular transformation. It has been shown that T Ag associates with an SCF-like complex that contains a member of the cullin family of E3 ubiquitin ligases, CUL7, as well as SKP1, RBX1, and an F-box protein, FBXW8. We identified T Ag residues 69 to 83 as required for T Ag binding to the CUL7 complex. We demonstrate that delta69-83 T Ag, while it lost its ability to associate with CUL7, retained binding to p53 and pRB family members. In the presence of CUL7, wild-type (WT) T Ag but not delta69-83 T Ag was able to induce proliferation of mouse embryo fibroblasts, an indication of cellular transformation. In contrast, WT and delta69-83 T Ag enabled mouse embryo fibroblasts to proliferate to similarly high densities in the absence of CUL7. Our data suggest that, in addition to p53 and the pRB family members, T Ag serves to bind to and inactivate the growth-suppressing properties of CUL7. In addition, these results imply that, at least in the presence of T Ag, CUL7 may function as a tumor suppressor.

Pheromone-dependent destruction of the Tec1 transcription factor is required for MAP kinase signaling specificity in yeast.

The yeast MAPK pathways required for mating versus filamentous growth share multiple components yet specify distinct programs. The mating-specific MAPK, Fus3, prevents crosstalk between the two pathways by unknown mechanisms. Here we show that pheromone signaling induces Fus3-dependent degradation of Tec1, the transcription factor specific to the filamentation pathway. Degradation requires Fus3 kinase activity and a MAPK phosphorylation site in Tec1 at threonine 273. Fus3 associates with Tec1 in unstimulated cells, and active Fus3 phosphorylates Tec1 on T273 in vitro. Destruction of Tec1 requires the F box protein Dia2 (Digs-into-agar-2), and Cdc53, the Cullin of SCF (Skp1-Cdc53-F box) ubiquitin ligases. Notably, mutation of the phosphoacceptor site in Tec1, deletion of FUS3, or deletion of DIA2 results in a loss of signaling specificity such that pheromone pathway signaling erroneously activates filamentation pathway gene expression and invasive growth. Signal-induced destruction of a transcription factor for a competing pathway provides a mechanism for signaling specificity.