The F-box Protein FBXO44 Mediates BRCA1 Ubiquitination and Degradation

BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCF^FBXO44) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCF^FBXO44 reduces BRCA1 protein level. Taken together, our work strongly suggests that SCF^FBXO44 is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCF^FBXO44-mediated BRCA1 degradation might contribute to sporadic breast tumor development.     

Sequences in the nonconsensus nucleotide-binding domain of ABCG5/ABCG8 required for sterol transport

ATP-binding cassette transporters ABCG5 (G5) and ABCG8 (G8) form a heterodimer that transports cholesterol and plant sterols from hepatocytes into bile. Mutations that inactivate G5 or G8 cause hypercholesterolemia and premature atherosclerosis. We showed previously that the two nucleotide-binding domains (NBDs) in the heterodimer are not functionally equivalent; sterol transport is abolished by mutations in the consensus residues of NBD2 but not of NBD1. Here, we examined the structural requirements of NBD1 for sterol transport. Substitutions of the D-loop aspartate and Q-loop glutamine in either NBD did not impair sterol transport. The H-loop histidine of NBD2 (but not NBD1) was required for sterol transport. Exchange of the signature motifs between the NBDs did not interfere with sterol transport, whereas swapping the Walker A, Walker B, and signature motifs together resulted in failure to transport sterols. Selected substitutions within NBD1 altered substrate specificity: transport of plant sterols by the heterodimer was preserved, whereas transport of cholesterol was abolished. In summary, these data indicate that NBD1, although not required for ATP hydrolysis, is essential for normal function of G5G8 in sterol transport. Both the position and structural integrity of NBD2 are essential for sterol transport activity.     

c-Cbl Facilitates Endocytosis and Lysosomal Degradation of Cystic Fibrosis Transmembrane Conductance Regulator in Human Airway Epithelial Cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl⁻ channel expressed in the apical membrane of fluid-transporting epithelia. The apical membrane density of CFTR channels is determined, in part, by endocytosis and the postendocytic sorting of CFTR for lysosomal degradation or recycling to the plasma membrane. Although previous studies suggested that ubiquitination plays a role in the postendocytic sorting of CFTR, the specific ubiquitin ligases are unknown. c-Cbl is a multifunctional molecule with ubiquitin ligase activity and a protein adaptor function. c-Cbl co-immunoprecipitated with CFTR in primary differentiated human bronchial epithelial cells and in cultured human airway cells. Small interfering RNA-mediated silencing of c-Cbl increased CFTR expression in the plasma membrane by inhibiting CFTR endocytosis and increased CFTR-mediated Cl⁻ currents. Silencing c-Cbl did not change the expression of the ubiquitinated fraction of plasma membrane CFTR. Moreover, the c-Cbl mutant with impaired ubiquitin ligase activity (FLAG-70Z-Cbl) did not affect the plasma membrane expression or the endocytosis of CFTR. In contrast, the c-Cbl mutant with the truncated C-terminal region (FLAG-Cbl-480), responsible for protein adaptor function, had a dominant interfering effect on the endocytosis and plasma membrane expression of CFTR. Moreover, CFTR and c-Cbl co-localized and co-immunoprecipitated in early endosomes, and silencing c-Cbl reduced the amount of ubiquitinated CFTR in early endosomes. In summary, our data demonstrate that in human airway epithelial cells, c-Cbl regulates CFTR by two mechanisms: first by acting as an adaptor protein and facilitating CFTR endocytosis by a ubiquitin-independent mechanism, and second by ubiquitinating CFTR in early endosomes and thereby facilitating the lysosomal degradation of CFTR.     

Lysosomal acid lipase deficiency impairs regulation of ABCA1 gene and formation of high density lipoproteins in cholesteryl ester storage disease

ATP-binding cassette transporter A1 (ABCA1) mediates the rate-limiting step in high density lipoprotein (HDL) particle formation, and its expression is regulated primarily by oxysterol-dependent activation of liver X receptors. We previously reported that ABCA1 expression and HDL formation are impaired in the lysosomal cholesterol storage disorder Niemann-Pick disease type C1 and that plasma HDL-C is low in the majority of Niemann-Pick disease type C patients. Here, we show that ABCA1 regulation and activity are also impaired in cholesteryl ester storage disease (CESD), caused by mutations in the LIPA gene that result in less than 5% of normal lysosomal acid lipase (LAL) activity. Fibroblasts from patients with CESD showed impaired up-regulation of ABCA1 in response to low density lipoprotein (LDL) loading, reduced phospholipid and cholesterol efflux to apolipoprotein A-I, and reduced α-HDL particle formation. Treatment of normal fibroblasts with chloroquine to inhibit LAL activity reduced ABCA1 expression and activity, similar to that of CESD cells. Liver X receptor agonist treatment of CESD cells corrected ABCA1 expression but failed to correct LDL cholesteryl ester hydrolysis and cholesterol efflux to apoA-I. LDL-induced production of 27-hydroxycholesterol was reduced in CESD compared with normal fibroblasts. Treatment with conditioned medium containing LAL from normal fibroblasts or with recombinant human LAL rescued ABCA1 expression, apoA-I-mediated cholesterol efflux, HDL particle formation, and production of 27-hydroxycholesterol by CESD cells. These results provide further evidence that the rate of release of cholesterol from late endosomes/lysosomes is a critical regulator of ABCA1 expression and activity, and an explanation for the hypoalphalipoproteinemia seen in CESD patients.     

Obesity and weight loss result in increased adipose tissue ABCG1 expression in db/db mice

The prevalence of obesity has reached epidemic proportions and is associated with several co-morbid conditions including diabetes, dyslipidemia, cancer, atherosclerosis and gallstones. Obesity is associated with low systemic inflammation and an accumulation of adipose tissue macrophages (ATMs) that are thought to modulate insulin resistance. ATMs may also modulate adipocyte metabolism and take up lipids released during adipocyte lipolysis and cell death. We suggest that high levels of free cholesterol residing in adipocytes are released during these processes and contribute to ATM activation and accumulation during obesity and caloric restriction. Db/db mice were studied for extent of adipose tissue inflammation under feeding conditions of ad libitum (AL) and caloric restriction (CR). The major finding was a marked elevation in epididymal adipose ABCG1 mRNA levels with obesity and CR (6-fold and 16-fold, respectively) over that seen for lean wild-type mice. ABCG1 protein was also elevated for CR as compared to AL adipose tissue. ABCG1 is likely produced by cholesterol loaded ATMs since this gene is not highly expressed in adipocytes and ABCG1 expression is sterol mediated. Our data supports the concept that metabolic changes in adipocytes due to demand lipolysis and cell death lead to cholesterol loading of ATMs. Based on finding cholesterol-loaded peritoneal leukocytes with elevated levels of ABCG1 in CR as compared to AL mice, we suggest that pathways for cholesterol trafficking out of adipose tissue involve ATM egress as well as ABCG1 mediated cholesterol efflux. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

LXR和ABCA1对体内胆固醇代谢的调节作用

肝外组织胆固醇返回肝脏,在肝脏通过生成胆汁酸排出,这一过程称为胆固醇逆转运。研究表明LXRs在维持体内胆固醇平衡方面起着感受器作用,通过关键靶基因转录的控制来调节胆固醇分解、储存、吸收和转运。LXR和RXR激动剂可上调巨噬细胞三磷酸腺苷结合盒转运体A1和G1(ABCAl,ABCGl)的表达,导致细胞内胆固醇流出。以LXR作为靶点的药物将为治疗高胆固醇血症和抗As提供新的希望。     

Caveolin-1 Interacts with Derlin-1 and Promotes Ubiquitination and Degradation of Cyclooxygenase-2 via Collaboration with p97 Complex

Caveolin-1 (Cav-1) interacts with and mediates protein trafficking and various cellular functions. Derlin-1 is a candidate for the retrotranslocation channel of endoplasmic reticulum proteins. However, little is known about how Derlin-1 mediates glycosylated protein degradation. Here, we identified Cav-1 as a key player in Derlin-1- and p97-mediated cyclooxygenase 2 (COX-2) ubiquitination and degradation. Derlin-1 augmented the interaction of Cav-1 and COX-2 and mediated the degradation of COX-2 in a COX-2 C terminus-dependent manner. Suppression of Cav-1 decreased the ubiquitination of COX-2, and mutation of Asn-594 to Ala to disrupt N-glycosylation at the C terminus of COX-2 reduced the interaction of COX-2 with Cav-1 but not Derlin-1. Moreover, suppression of p97 increased the ubiquitination of COX-2 and up-regulated COX-2 but not COX-1. Cav-1 enhanced the interaction of p97 with Ufd1 and Derlin-1 and collaborated with p97 to interact with COX-2. Cav-1 may be a cofactor in the interaction of Derlin-1 and N-glycosylated COX-2 and may facilitate Derlin-1- and p97 complex-mediated COX-2 ubiquitination, retrotranslocation, and degradation.     

The ABCA1 transporter functions on the basolateral surface of hepatocytes

ABCA1 on the cell surface and in endosomes plays an essential role in the cell-mediated lipidation of apoA-I to form nascent HDL. Our previous studies of transgenic mice overexpressing ABCA1 suggested that ABCA1 in the liver plays a major role in regulating plasma HDL levels. The site of function of ABCA1 in the polarized hepatocyte was currently assessed by expression of an adenoviral construct encoding a human ABCA1-GFP fusion protein in the polarized hepatocyte-like WIF-B cell line. Consistent with localization of ABCA1 at the basolateral (vascular) cell surface, expression of ABCA1-GFP stimulated apoA-I mediated efflux of WIF-B cell cholesterol into the culture medium. Confocal fluorescence microscopy revealed that ABCA1-GFP was expressed solely on the basolateral surface and associated endocytic vesicles. These findings suggest an important role for hepatocyte basolateral membrane ABCA1 in the regulation of the levels of intracellular hepatic cholesterol, as well as plasma HDL.     

ASK1 Negatively Regulates the 26 S Proteasome

The 26 S proteasome, composed of the 20 S core and 19 S regulatory particle, plays a central role in ubiquitin-dependent proteolysis. Disruption of this process contributes to the pathogenesis of the various diseases; however, the mechanisms underlying the regulation of 26 S proteasome activity remain elusive. Here, cell culture experiments and in vitro assays demonstrated that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPK kinase kinase family, negatively regulated 26 S proteasome activity. Immunoprecipitation/Western blot analyses revealed that ASK1 did not interact with 20 S catalytic core but did interact with ATPases making up the 19 S particle, which is responsible for recognizing polyubiquitinated proteins, unfolding them, and translocating them into the 20 S catalytic core in an ATP-dependent process. Importantly, ASK1 phosphorylated Rpt5, an AAA ATPase of the 19 S proteasome, and inhibited its ATPase activity, an effect that may underlie the ability of ASK1 to inhibit 26 S proteasome activity. The current findings point to a novel role for ASK1 in the regulation of 26 S proteasome and offer new strategies for treating human diseases caused by proteasome malfunction.     

The E3 ubiquitin ligase,HECTD1, is involved in ABCA1-mediated cholesterol export from macrophages

The ABC lipid transporters, ABCA1 and ABCG1, are essential for maintaining lipid homeostasis in cells such as macrophages by exporting excess cholesterol to extracellular acceptors. These transporters are highly regulated at the post-translational level, including protein ubiquitination. Our aim was to investigate the role of the E3 ubiquitin ligase HECTD1, recently identified as associated with ABCG1, on ABCG1 and ABCA1 protein levels and cholesterol export function. Here, we show that HECTD1 protein is widely expressed in a range of human and murine primary cells and cell lines, including macrophages, neuronal cells and insulin secreting β-cells. siRNA knockdown of HECTD1 unexpectedly decreased overexpressed ABCG1 protein levels and cell growth, but increased native ABCA1 protein in CHO-K1 cells. Knockdown of HECTD1 in unloaded THP-1 macrophages did not affect ABCG1 but significantly increased ABCA1 protein levels, in wild-type as well as THP-1 cells that do not express ABCG1. Cholesterol export from macrophages to apoA-I over time was increased after knockdown of HECTD1, however these effects were not sustained in cholesterol-loaded cells. In conclusion, we have identified a new candidate, the E3 ubiquitin ligase HECTD1, that may be involved in the regulation of ABCA1-mediated cholesterol export from unloaded macrophages to apoA-I. The exact mechanism by which this ligase affects this pathway remains to be elucidated.     

Molecular Mechanisms of Cellular Cholesterol Efflux

Most types of cells in the body do not express the capability of catabolizing cholesterol, so cholesterol efflux is essential for homeostasis. For instance, macrophages possess four pathways for exporting free (unesterified) cholesterol to extracellular high density lipoprotein (HDL). The passive processes include simple diffusion via the aqueous phase and facilitated diffusion mediated by scavenger receptor class B, type 1 (SR-BI). Active pathways are mediated by the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, which are membrane lipid translocases. The efflux of cellular phospholipid and free cholesterol to apolipoprotein A-I promoted by ABCA1 is essential for HDL biogenesis. Current understanding of the molecular mechanisms involved in these four efflux pathways is presented in this minireview.     

转运蛋白ABCG1/4 和ABCA1对脑胆固醇的调节作用

脑是富含胆固醇的器官,机体大约有25%的胆固醇集中在脑组织中.ATP结合盒超家族转运蛋白对脑组织中胆固醇的膜外转运和动态平衡起着重要的调节作用.研究发现,ATP结合盒超家族转运蛋白亚体ABCG1、ABCG4和ABCA1在成体脑组织中存在不同程度的表达,一种或多种亚体的缺失可以导致神经退行性病变.然而,ATP结合盒超家族转运蛋白亚体对脑发育过程中脑胆固醇动态变化的调节缺乏相关性的报道.在本研究中,从低胆固醇饮食喂养的C57BL/6J小鼠中获取出生后不同发育时期的脑组织,对ABCG1、ABCG4和ABCA1的mRNA与蛋白质表达水平进行测定,并对脑组织和血清中ATP结合盒超家族转运蛋白的表达水平与胆固醇水平的相关性进行研究.同时,使用ABCG1、ABCG4单一基因敲除鼠和ABCG1、ABCG4双基因敲除鼠,研究ATP结合盒超家族转运蛋白对与胆固醇合成的相关基因表达的影响以及对脑组织胆固醇代谢的调节作用.结果发现,ABCG1、ABCG4和ABCA1在机体多个器官中均有表达,但ABCG1和ABCG4在小鼠脑组织中表达量最高.在脑组织发育过程中,ABCG1和ABCG4mRNA水平呈现明显的表达时效性,小鼠于出生后42天达到峰值,而ABCA1 mRNA的表达水平无明显变化.血清和脑组织中中酯化型胆固醇水平呈双高峰分布,也于出生后42天达到最高.基因敲除鼠模型显示,单一敲除ABCG1或者ABCG4基因对脑组织胆固醇水平无明显影响,而ABCG1和ABCG4基因的同时缺失导致脑胆固醇水平显著升高,并明显降低胆固醇合成相关基因的表达水平.本研究表明,在脑发育成熟过程中,ATP结合盒超家族转运蛋白亚体ABCG1和ABCG4,而非ABCA1,以调节脑胆固醇的膜外转运;ABCG1和ABCG4互补调控脑胆固醇的动态平衡.     

24S-hydroxycholesterol induces cholesterol release from choroid plexus epithelial cells in an apical- and apoE isoform-dependent manner concomitantly with the induction of ABCA1 and ABCG1 expression

The release of cholesterol from choroid plexus epithelial cells (CPE) plays an important role in cholesterol homeostasis in the CSF. The purpose of this study was to clarify the molecules involved in cholesterol release in CPE and the regulation mechanisms of the cholesterol release by the liver X receptor (LXR) using a conditionally immortalized CPE line (TR-CSFB3). The mRNA expression of LXRalpha, LXRbeta and their target genes, ATP-binding cassette transporter (ABC)A1, ABCG1, ABCG4 and ABCG5, were detected in rat choroid plexus. ABCA1 and ABCG1 protein were detected in the plasma membrane of TR-CSFB3 cells. Following treatment with 24S-hydroxycholesterol, an endogenous LXR ligand, the expression of ABCA1 and ABCG1 were induced in TR-CSFB3 cells. Moreover, apolipoprotein (apo)AI- and high-density lipoprotein (HDL)-mediated cholesterol release to the apical side of TR-CSFB3 cells was facilitated by this treatment, whereas that to the basal side was not affected. Following 24S-hydroxycholesterol treatment, apoE3-dependent cholesterol release from TR-CSFB3 cells was enhanced more than the apoE4-dependent release. These results suggest that LXR activation facilitates cholesterol release into the CSF from CPE through the functional induction of ABCA1 and ABCG1. The difference between apoE3 and apoE4 suggests that the cholesterol release from CPE is related to the development of neurodegenerative diseases.     

24(S)‐hydroxycholesterol is actively eliminated from neuronal cells by ABCA1

High cholesterol turnover catalyzed by cholesterol 24‐hydroxylase is essential for neural functions, especially learning. Because 24(S)‐hydroxycholesterol (24‐OHC), produced by 24‐hydroxylase, induces apoptosis of neuronal cells, it is vital to eliminate it rapidly from cells. Here, using differentiated SH‐SY5Y neuron‐like cells as a model, we examined whether 24‐OHC is actively eliminated via transporters induced by its accumulation. The expression of ABCA1 and ABCG1 was induced by 24‐OHC, as well as TO901317 and retinoic acid, which are ligands of the nuclear receptors liver X receptor/retinoid X receptor (LXR/RXR). When the expression of ABCA1 and ABCG1 was induced, 24‐OHC efflux was stimulated in the presence of high‐density lipoprotein (HDL), whereas apolipoprotein A‐I was not an efficient acceptor. The efflux was suppressed by the addition of siRNA against ABCA1, but not by ABCG1 siRNA. To confirm the role of each transporter, we analyzed human embryonic kidney 293 cells stably expressing human ABCA1 or ABCG1; we clearly observed 24‐OHC efflux in the presence of HDL, whereas efflux in the presence of apolipoprotein A‐I was marginal. Furthermore, the treatment of primary cerebral neurons with LXR/RXR ligands suppressed the toxicity of 24‐OHC. These results suggest that ABCA1 actively eliminates 24‐OHC in the presence of HDL as a lipid acceptor and protects neuronal cells.     

Functional expression and characterization of the human ABCG1 and ABCG4 proteins: indications for heterodimerization

The closely related human ABC half-transporters, ABCG1 and ABCG4, have been suggested to play an important role in cellular lipid/sterol regulation but no experimental data for their expression or function are available. We expressed ABCG1 and ABCG4 and their catalytic site mutant variants in insect cells, generated specific antibodies, and analyzed their function in isolated membrane preparations. ABCG1 had a high basal ATPase activity, further stimulated by lipophilic cations and significantly inhibited by cyclosporin A, thyroxine or benzamil. ABCG4 had a lower basal ATPase activity which was not modulated by any of the tested compounds. The catalytic site (K-M) mutants had no ATPase activity. Since dimerization is a requirement for half-transporters, we suggest that both ABCG1 and ABCG4 function as homodimers. Importantly, we also found that co-expression of the ABCG4-KM mutant selectively abolished the ATPase activity of the ABCG1 and therefore they most probably also heterodimerize. The heterologous expression, specific recognition, and functional characterization of these transporters should help to delineate their physiological role and mechanism of action.     

Identification of the Degradation Determinants of Insulin Receptor Substrate 1 for Signaling Cullin-RING E3 Ubiquitin Ligase 7-mediated Ubiquitination

Hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and its effector kinase S6 kinase 1 (S6K1) is known to trigger multisite seryl phosphorylation of insulin receptor substrate 1 (IRS1), leading to its ubiquitination and degradation. This negative feedback inhibition functions to restrain PI3K activity and plays critical roles in the pathogenesis of cancer and type II diabetes. Recent work has implicated a role for cullin-RING E3 ubiquitin ligase 7 (CRL7) in targeting IRS1 for mTORC1/S6K1-dependent degradation. In the present study we have employed both cell-based degradation and reconstituted ubiquitination approaches to define molecular features associated with IRS1 critical for CRL7-mediated ubiquitination and degradation. We have mapped IRS1 degradation signal sequence to its N-terminal 574 amino acid residues, of which the integrity of Ser-307/Ser-312 and Ser-527, each constituting a S6K1 phosphorylation consensus site, was indispensible for supporting CRL7-forced degradation. In vitro, S6K1 was able to support the ubiquitination of bacterially expressed IRS1 N-terminal fragment by CRL7 but at low levels. In contrast, CRL7 supported efficient ubiquitination of IRS1 N-terminal fragment in hyperphosphorylated form, which was isolated from infected insect cells, suggesting requirement of additional phosphorylation by kinases yet to be identified. Finally, removal of IRS1 amino acids 1–260 led to substantial reduction of ubiquitination efficiency, suggesting a role for this region in mediating productive interactions with CRL7. The requirement of multisite phosphorylation and the N terminus of IRS1 for its turnover may ensure that complete IRS1 degradation occurs only when mTORC1 and S6K1 reach exceedingly high levels.