Effects of UBE3A on Cell and Liver Metabolism through the Ubiquitination of PDHA1 and ACAT1.

Nonalcoholic fatty liver disease (NAFLD) is substantiated by the reprogramming of liver metabolic pathways that disrupts the homeostasis of lipid and glucose metabolism and thus promotes the progression of the disease. The metabolic pathways associated with NAFLD are regulated at different levels from gene transcription to various post-translational modifications including ubiquitination. Here, we used a novel orthogonal ubiquitin transfer platform to identify pyruvate dehydrogenase A1 (PDHA1) and acetyl-CoA acetyltransferase 1 (ACAT1), two important enzymes that regulate glycolysis and ketogenesis, as substrates of E3 ubiquitin ligase UBE3A/E6AP. We found that overexpression of UBE3A accelerated the degradation of PDHA1 and promoted glycolytic activities in HEK293 cells. Furthermore, a high-fat diet suppressed the expression of UBE3A in the mouse liver, which was associated with increased ACAT1 protein levels, while forced expression of UBE3A in the mouse liver resulted in decreased ACAT1 protein contents. As a result, the mice with forced expression of UBE3A in the liver exhibited enhanced accumulation of triglycerides, cholesterol, and ketone bodies. These results reveal the role of UBE3A in NAFLD development by inducing the degradation of ACAT1 in the liver and promoting lipid storage. Overall, our work uncovers an important mechanism underlying the regulation of glycolysis and lipid metabolism through UBE3A-mediated ubiquitination of PDHA1 and ACAT1 to regulate their stabilities and enzymatic activities in the cell.

Effects of specular roughness on the perception of color and opacity.

Previous studies have shown that information concerning object shape is important for the perception of translucency. This study aims to explore how the perception of semi-opaque objects is influenced by surface gloss. We varied specular roughness, specular amplitude, and the simulated direction of a light source used to illuminate a globally convex bumpy object. We found that perceived lightness and roughness increased as specular roughness was increased. Declines in perceived saturation were observed but were far smaller in magnitude with these increases in specular roughness. There were inverse correlations found between perceived gloss and perceived lightness, perceived transmittance and perceived saturation, and between perceived roughness and perceived gloss. Positive correlations were found between perceived transmittance and glossiness, and between perceived roughness and perceived lightness. These findings suggest that specular reflections influence the perception of transmittance and color attributes, and not just perceived gloss. We also performed follow-up modeling of image data to find that perceived saturation and lightness could be explained by the reliance on different image regions with greater chroma and lower lightness, respectively. We also found systematic effects of lighting direction on perceived transmittance that indicate there are complex perceptual interactions that require further consideration.

Orthogonal ubiquitin transfer reveals human papillomavirus E6 downregulates nuclear transport to disarm interferon-γ dependent apoptosis of cervical cancer cells.

The E6 protein of the human papillomavirus (HPV) underpins important protein interaction networks between the virus and host to promote viral infection. Through its interaction with E6AP, a host E3 ubiquitin (UB) ligase, E6 stirs the protein ubiquitination pathways toward the oncogenic transformation of the infected cells. For a systematic measurement of E6 reprogramming of the substrate pool of E6AP, we performed a proteomic screen based on "orthogonal UB transfer (OUT)" that allowed us to identify the ubiquitination targets of E6AP dependent on the E6 protein of HPV-16, a high-risk viral subtype for the development of cervical cancer. The OUT screen identified more than 200 potential substrates of the E6-E6AP pair based on the transfer of UB from E6AP to the substrate proteins. Among them, we verified that E6 would induce E6AP-catalyzed ubiquitination of importin proteins KPNA1-3, protein phosphatase PGAM5, and arginine methyltransferases CARM1 to trigger their degradation by the proteasome. We further found that E6 could significantly reduce the cellular level of KPNA1 that resulted in the suppression of nuclear transport of phosphorylated STAT1 and the inhibition of interferon-γ-induced apoptosis in cervical cancer cells. Overall, our work demonstrates OUT as a powerful proteomic platform to probe the interaction of E6 and host cells through protein ubiquitination and reveals a new role of E6 in down-regulating nuclear transport proteins to attenuate tumor-suppressive signaling.

Regulation of O-Linked N-Acetyl Glucosamine Transferase (OGT) through E6 Stimulation of the Ubiquitin Ligase Activity of E6AP.

Glycosyltransferase OGT catalyzes the conjugation of O-linked ß-D-N-acetylglucosamine (O-GlcNAc) to Ser and Thr residues of the cellular proteins and regulates many key processes in the cell. Here, we report the identification of OGT as a ubiquitination target of HECT-type E3 ubiquitin (UB) ligase E6AP, whose overexpression in HEK293 cells would induce the degradation of OGT. We also found that the expression of E6AP in HeLa cells with the endogenous expression of the E6 protein of the human papillomavirus (HPV) would accelerate OGT degradation by the proteasome and suppress O-GlcNAc modification of OGT substrates in the cell. Overall, our study establishes a new mechanism of OGT regulation by the ubiquitin-proteasome system (UPS) that mediates the crosstalk between protein ubiquitination and O-GlcNAcylation pathways underlying diverse cellular processes.

Luminance edge is a cue for glossiness perception based on low-luminance specular components.

The visual system is considered to employ various image cues from an object image to perceive its glossiness. It has been reported that, surprisingly, even for object images without specular highlights we can perceive glossiness by relying on low-luminance specular components (Kim, Marlow, & Anderson, 2012). This type of perceptual glossiness is referred to as dark gloss. However, it is still unclear whether dark gloss is observed commonly across various objects, and what image features are cues for dark gloss. To address these issues, we performed several psychophysical experiments. First, we measured perceived glossiness for a number of computer-graphics object images with natural specular reflection components (Full condition) and for those without high-luminance components of specular reflections (Dark condition). The results showed that dark gloss (glossiness perception in the Dark condition) was generally observed on almost all object images, while its intensity was rather different across the images. Then we psychologically or computationally measured several image features for the stimulus images, such as luminance edge number, recognizability of reflection images, and some highlight-related features, to examine their relations to perceived glossiness with a multiple regression analysis. The results demonstrated that luminance edge number was most strongly related to glossiness scores among the measured features only for object images with potent dark gloss. These results suggest that luminance edges are an effective cue for dark gloss under certain stimulus conditions.

SUMO-mimicking peptides inhibiting protein SUMOylation.

The ubiquitin-like protein SUMO is transferred through a core E1-E2 cascade composed of the SUMO-activating enzyme (SAE) and Ubc9 to modify cellular proteins and transmit important biological signals. SAE primarily recognizes the C-terminal tail of SUMO and catalyzes ATP condensation with the SUMO C-terminal carboxylate to activate its transfer through the cascade. Here, we used phage display to show that a broad profile of SUMO C-terminal sequences could be activated by SAE. Based on this, we developed heptamer peptides that could 1) form thioester conjugates with SAE, 2) be transferred from SAE to Ubc9, and 3) be further transferred to the SUMOylation target protein RanGAP1. As these peptides recapitulate the action of SUMO in protein modification, we refer to them as "SUMO-mimicking peptides". We found that, once the peptides were conjugated to SAE and Ubc9, they blocked full-length SUMO from entering the cascade. These peptides can thus function as mechanism-based inhibitors of the protein SUMOylation reaction.

Downregulation of Smurf2, a tumor-suppressive ubiquitin ligase, in triple-negative breast cancers: involvement of the RB-microRNA axis.

BACKGROUND: The HECT family ubiquitin ligase Smurf2 regulates cell polarity, migration, division, differentiation and death, by targeting diverse substrates that are critical for receptor signaling, cytoskeleton, chromatin remodeling and transcription. Recent studies suggest that Smurf2 functions as a tumor suppressor in mice. However, no inactivating mutation of SMURF2 has been reported in human, and information about Smurf2 expression in human cancer remains limited or complicated. Here we demonstrate that Smurf2 expression is downregulated in human breast cancer tissues, especially of the triple-negative subtype, and address the mechanism of Smurf2 downregulation in triple-negative breast cancer cells. METHODS: Human breast cancer tissues (47 samples expressing estrogen receptor (ER) and 43 samples with triple-negative status) were examined by immunohistochemistry for the expression of Smurf2. Ten widely-studied human breast cancer cell lines were examined for the expression of Smurf2. Furthermore, microRNA-mediated regulation of Smurf2 was investigated in triple-negative cancer cell lines. RESULTS: Immunohistochemical analysis showed that benign mammary epithelial cells expressed high levels of Smurf2, so did cells in ductal carcinomas in situ. In contrast, invasive ductal carcinomas showed focal or diffuse decrease in Smurf2 expression, which was observed more frequently in triple-negative tumors than in ER-positive tumors. Consistently, human triple-negative breast cancer cell lines such as BT549, MDA-MB-436, DU-4475 and MDA-MB-468 cells showed significantly lower expression of Smurf2 protein, compared to ER + or HER2+ cell lines. Studies using quantitative PCR and specific microRNA inhibitors indicated that increased expression of miR-15a, miR-15b, miR-16 and miR-128 was involved in Smurf2 downregulation in those triple-negative cancer cell lines, which have mutations in the retinoblastoma (RB) gene. Forced expression of RB increased levels of Smurf2 protein with concomitant decreases in the expression of the microRNAs. CONCLUSIONS: This study provides evidence of posttranscriptional downregulation of Smurf2 in triple-negative breast cancers, and demonstrates that the loss of RB function is involved in microRNA-mediated interference with Smurf2 translation. The new link from RB inactivation to Smurf2 downregulation is likely to play a role in malignant phenotypes of triple-negative breast cancer cells.

Akt deficiency impairs normal cell proliferation and suppresses oncogenesis in a p53-independent and mTORC1-dependent manner.

Akt contributes to tumorigenesis by inhibiting apoptosis. Here we establish that Akt is required for normal cell proliferation and susceptibility to oncogenesis independently of its antiapoptotic activity. Partial ablation of Akt activity by deleting Akt1 inhibits cell proliferation and oncogenesis. These effects are compounded by deleting both Akt1 and Akt2. In vivo, Akt1 null mice are resistant to MMTV-v-H-Ras-induced tumors and to skin carcinogenesis. Thus, partial ablation of Akt activity is sufficient to suppress tumorigenesis in vitro and in vivo. The effect of Akt deficiency on cell proliferation and oncogenesis is p53 independent but mTORC1 dependent. Surprisingly, upon mTORC1 hyperactivation, the reduction in Akt activity does not impair cell proliferation and susceptibility to oncogenic transformation; thus, Akt may mediate these processes exclusively via mTORC1.

Requirement for CDK4 kinase function in breast cancer.

Cyclin D1 is overexpressed in the majority of human breast cancers. We previously found that mice lacking cyclin D1 are resistant to mammary carcinomas triggered by the ErbB-2 oncogene. In this study, we investigated which function of cyclin D1 is required for ErbB-2-driven mammary oncogenesis. We report that the ability of cyclin D1 to activate cyclin-dependent kinase CDK4 underlies the critical role for cyclin D1 in breast cancer formation. We also found that the continued presence of CDK4-associated kinase activity is required to maintain breast tumorigenesis. We analyzed primary human breast cancers and found high cyclin D1 levels in a subset (approximately 25%) of ErbB-2-overexpressing tumors. We propose that this subset of breast cancer patients might benefit from inhibiting CDK4 kinase.

Eukaryotic initiation factor 6 is rate-limiting in translation, growth and transformation.

Cell growth and proliferation require coordinated ribosomal biogenesis and translation. Eukaryotic initiation factors (eIFs) control translation at the rate-limiting step of initiation. So far, only two eIFs connect extracellular stimuli to global translation rates: eIF4E acts in the eIF4F complex and regulates binding of capped messenger RNA to 40S subunits, downstream of growth factors, and eIF2 controls loading of the ternary complex on the 40S subunit and is inhibited on stress stimuli. No eIFs have been found to link extracellular stimuli to the activity of the large 60S ribosomal subunit. eIF6 binds 60S ribosomes precluding ribosome joining in vitro. However, studies in yeasts showed that eIF6 is required for ribosome biogenesis rather than translation. Here we show that mammalian eIF6 is required for efficient initiation of translation, in vivo. eIF6 null embryos are lethal at preimplantation. Heterozygous mice have 50% reduction of eIF6 levels in all tissues, and show reduced mass of hepatic and adipose tissues due to a lower number of cells and to impaired G1/S cell cycle progression. eIF6(+/-) cells retain sufficient nucleolar eIF6 and normal ribosome biogenesis. The liver of eIF6(+/-) mice displays an increase of 80S in polysomal profiles, indicating a defect in initiation of translation. Consistently, isolated hepatocytes have impaired insulin-stimulated translation. Heterozygous mouse embryonic fibroblasts recapitulate the organism phenotype and have normal ribosome biogenesis, reduced insulin-stimulated translation, and delayed G1/S phase progression. Furthermore, eIF6(+/-) cells are resistant to oncogene-induced transformation. Thus, eIF6 is the first eIF associated with the large 60S subunit that regulates translation in response to extracellular signals.

Inhibition of Cdc25A suppresses hepato-renal cystogenesis in rodent models of polycystic kidney and liver disease.

BACKGROUND & AIMS: In polycystic kidney disease and polycystic liver disease (PLD), the normally nonproliferative hepato-renal epithelia acquire a proliferative, cystic phenotype that is linked to overexpression of cell division cycle 25 (Cdc25)A phosphatase and cell-cycle deregulation. We investigated the effects of Cdc25A inhibition in mice and rats via genetic and pharmacologic approaches. METHODS: Cdc25A(+/-) mice (which have reduced levels of Cdc25A) were cross-bred with polycystic kidney and hepatic disease 1 (Pkhd1(del2/del2)) mice (which have increased levels of Cdc25A and develop hepatic cysts). Cdc25A expression was analyzed in livers of control and polycystic kidney (PCK) rats, control and polycystic kidney 2 (Pkd2(ws25/-)) mice, healthy individuals, and patients with PLD. We examined effects of pharmacologic inhibition of Cdc25A with vitamin K3 (VK3) on the cell cycle, proliferation, and cyst expansion in vitro; hepato-renal cystogenesis in PCK rats and Pkd2(ws25/-)mice; and expression of Cdc25A and the cell-cycle proteins regulated by Cdc25A. We also examined the effects of the Cdc25A inhibitor PM-20 on hepato-renal cystogenesis in Pkd2(ws25/-) mice. RESULTS: Liver weights and hepatic and fibrotic areas were decreased by 32%-52% in Cdc25A(+/-):Pkhd1(del2/del2) mice, compared with Pkhd1(del2/del2) mice. VK3 altered the cell cycle and reduced proliferation of cultured cholangiocytes by 32%-83% and decreased growth of cultured cysts by 23%-67%. In PCK rats and Pkd2(ws25/-) mice, VK3 reduced liver and kidney weights and hepato-renal cystic and fibrotic areas by 18%-34%. PM-20 decreased hepato-renal cystogenesis in Pkd2(ws25/-) mice by 15%. CONCLUSIONS: Cdc25A inhibitors block cell-cycle progression and proliferation, reduce liver and kidney weights and cyst growth in animal models of polycystic kidney disease and PLD, and might be developed as therapeutics for these diseases.

Phage display to identify Nedd8-mimicking peptides as inhibitors of the Nedd8 transfer cascade.

The Nedd8 activating enzyme (NAE) launches the transfer of the ubiquitin-like protein Nedd8 through an enzymatic cascade to covalently modify a diverse array of proteins, thus regulating their biological functions in the cell. The C-terminal peptide of Nedd8 extends deeply into the active site of NAE and plays an important role in the specific recognition of Nedd8 by NAE. We used phage display to profile C-terminal mutant sequences of Nedd8 that could be recognized by NAE for the activation reaction. We found that NAE can accommodate diverse changes in the Nedd8 C-terminal sequence (7¹ LALRGG76), including Arg and Ile replacing Leu71, Leu, Ser, and Gln replacing Ala72, and substitutions by bulky aromatic residues at positions 73 and 74. We also observed that short peptides corresponding to the C-terminal sequences of the Nedd8 variants can be activated by NAE to form peptide~NAE thioester conjugates. Once NAE is covalently loaded with these Nedd8-mimicking peptides, it can no longer activate full-length Nedd8 for transfer to the neddylation targets, such as the cullin subunits of cullin-RING E3 ubiquitin ligases (CRLs). We have thus developed a new method to inhibit protein neddylation by Nedd8-mimicking peptides.

The perception of translucency from surface gloss.

Translucent objects (like fruit and wax) reflect and transmit incident light to generate complex retinal image structure. Understanding how we visually perceive translucency from these images is challenging, but previous studies have demonstrated that perceived shape and shading is important for perceiving translucency. We considered the possibility that perceived translucency might also depend on 3D shape inferred from surface gloss (i.e., shape from specular highlights). Here, we performed experiments to test whether interactions between specular and non-specular image properties generated by different 3D shape information influences perceived translucency. Results revealed that perceived translucency could be explained by incongruence in 3D shape used to generate specular and non-specular image components. We proposed a new computational model based on measurable image features informative of shading relative to specular highlights that accounted for 59% of the variability in judgments of perceived translucency from the result of 10-fold cross validation. This model was found to outperform other models based on explicit subjective measures of perceived surface shape, suggesting it implicitly taps much of the relevant geometric information necessary for predicting observer judgments of translucency for glossy materials. These results provide new insight into how the visual system might infer translucency from the structure of specular and non-specular shading generated by glossy semi-opaque materials.

Profiling and verifying the substrates of E3 ubiquitin ligase Rsp5 in yeast cells.

Yeast is an essential model organism for studying protein ubiquitination pathways; however, identifying the direct substrates of E3 in the cell presents a challenge. Here, we present a protocol for using the orthogonal ubiquitin transfer (OUT) cascade to profile the substrate specificity of yeast E3 Rsp5. We describe steps for OUT profiling, proteomics analysis, in vitro and in cell ubiquitination, and stability assay. The protocol can be adapted for identifying and verifying the ubiquitination targets of other E3s in yeast. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

Cdk6-dependent regulation of G(1) length controls adult neurogenesis.

The presence of neurogenic precursors in the adult mammalian brain is now widely accepted, but the mechanisms coupling their proliferation with the onset of neuronal differentiation remain unknown. Here, we unravel the major contribution of the G(1) regulator cyclin-dependent kinase 6 (Cdk6) to adult neurogenesis. We found that Cdk6 was essential for cell proliferation within the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricles. Specifically, Cdk6 deficiency prevents the expansion of neuronally committed precursors by lengthening G(1) phase duration, reducing concomitantly the production of newborn neurons. Altogether, our data support G(1) length as an essential regulator of the switch between proliferation and neuronal differentiation in the adult brain and Cdk6 as one intrinsic key molecular regulator of this process.

Cdc2-cyclin E complexes regulate the G1/S phase transition.

The cyclin-dependent kinase inhibitor p27(Kip1) is known as a negative regulator of cell-cycle progression and as a tumour suppressor. Cdk2 is the main target of p27 (refs 2, 3) and therefore we hypothesized that loss of Cdk2 activity should modify the p27(-/-) mouse phenotype. Here, we show that although p27(-/-) Cdk2(-/-) mice developed ovary tumours and tumours in the anterior lobe of the pituitary, we failed to detect any functional complementation in p27(-/-) Cdk2(-/-) double-knockout mice, indicating a parallel pathway regulated by p27. We observed elevated levels of S phase and mitosis in tissues of p27(-/-) Cdk2(-/-) mice concomitantly with elevated Cdc2 activity in p27(-/-) Cdk2(-/-) extracts. p27 binds to Cdc2, cyclin B1, cyclin A2, or suc1 complexes in wild-type and Cdk2(-/-) extracts. In addition, cyclin E binds to and activates Cdc2. Our in vivo results provide strong evidence that Cdc2 may compensate the loss of Cdk2 function.

Combined loss of Cdk2 and Cdk4 results in embryonic lethality and Rb hypophosphorylation.

Mouse knockouts of Cdk2 and Cdk4 have demonstrated that, individually, these genes are not essential for viability. To investigate whether there is functional redundancy, we have generated double knockout (DKO) mice. Cdk2-/- Cdk4-/- DKOs die during embryogenesis around E15 as a result of heart defects. We observed a gradual decrease of Retinoblastoma protein (Rb) phosphorylation and reduced expression of E2F-target genes, like Cdc2 and cyclin A2, during embryogenesis and in embryonic fibroblasts (MEFs). DKO MEFs are characterized by a decreased proliferation rate, impaired S phase entry, and premature senescence. HPV-E7-mediated inactivation of Rb restored normal expression of E2F-inducible genes, senescence, and proliferation in DKO MEFs. In contrast, loss of p27 did not rescue Cdk2-/- Cdk4-/- phenotypes. Our results demonstrate that Cdk2 and Cdk4 cooperate to phosphorylate Rb in vivo and to couple the G1/S phase transition to mitosis via E2F-dependent regulation of gene expression.

Spatial Frequency Effective for Increasing Perceived Glossiness by Contrast Enhancement.

It has been suggested that luminance edges in retinal images are potential cues for glossiness perception, particularly when the perception relies on low-luminance specular regions. However, a previous study has shown only statistical correlations between luminance edges and perceived glossiness, not their causal relations. Additionally, although specular components should be embedded at various spatial frequencies depending on the micro-roughness on the object surface, it is not well understood what spatial frequencies are essential for glossiness perception on objects with different micro-roughness. To address these issues, we examined the impact of a sub-band contrast enhancement on the perceived glossiness in the two conditions of stimuli: the Full condition where the stimulus had natural specular components and the Dark condition where it had specular components only in dark regions. Object images with various degrees of surface roughness were generated as stimuli, and their contrast was increased in various spatial-frequency sub-bands. The results indicate that the enhancement of the sub-band contrast can significantly increase perceived glossiness as expected. Furthermore, the effectiveness of each spatial frequency band depends on the surface roughness in the Full condition. However, effective spatial frequencies are constant at a middle spatial frequency regardless of the stimulus surface roughness in the Dark condition. These results suggest that, for glossiness perception, our visual system depends on specular-related information embedded in high spatial frequency components but may change the dependency on spatial frequency based on the surface luminance to be judged.

Regulation of the endocytosis and prion-chaperoning machineries by yeast E3 ubiquitin ligase Rsp5 as revealed by orthogonal ubiquitin transfer.

Attachment of the ubiquitin (UB) peptide to proteins via the E1-E2-E3 enzymatic machinery regulates diverse biological pathways, yet identification of the substrates of E3 UB ligases remains a challenge. We overcame this challenge by constructing an "orthogonal UB transfer" (OUT) cascade with yeast E3 Rsp5 to enable the exclusive delivery of an engineered UB (xUB) to Rsp5 and its substrate proteins. The OUT screen uncovered new Rsp5 substrates in yeast, such as Pal1 and Pal2, which are partners of endocytic protein Ede1, and chaperones Hsp70-Ssb, Hsp82, and Hsp104 that counteract protein misfolding and control self-perpetuating amyloid aggregates (prions), resembling those involved in human amyloid diseases. We showed that prion formation and effect of Hsp104 on prion propagation are modulated by Rsp5. Overall, our work demonstrates the capacity of OUT to deconvolute the complex E3-substrate relationships in crucial biological processes such as endocytosis and protein assembly disorders through protein ubiquitination.

Cdk4 promotes adipogenesis through PPARgamma activation.

Cell cycle regulators such as E2F1 and retinoblastoma (RB) play crucial roles in the control of adipogenesis, mostly by controlling the transition between preadipocyte proliferation and adipocyte differentiation. The serine-threonine kinase cyclin-dependent kinase 4 (cdk4) works in a complex with D-type cyclins to phosphorylate RB, mediating the entry of cells into the cell cycle in response to external stimuli. Because cdk4 is an upstream regulator of the E2F-RB pathway, we tested whether cdk4 was a target for new factors that regulate adipogenesis. Here we find that cdk4 inhibition impairs adipocyte differentiation and function. Disruption of cdk4 or activating mutations in cdk4 in primary mouse embryonic fibroblasts results in reduced and increased adipogenic potential, respectively, of these cells. We show that the effects of cdk4 are not limited to the control of differentiation; cdk4 also participates in adipocyte function through activation of PPARgamma.