Enhanced Protein Degradation Research Articles

Hydrophobic statins induce autophagy and cell death in human rhabdomyosarcoma cells by depleting geranylgeranyl diphosphate

Statins are the most common type of medicine used to treat hypercholesterolemia; however, they are associated with a low incidence of myotoxicity such as myopathy and rhabdomyolysis. The mechanisms for the adverse effects remain to be fully elucidated for safer chronic use and drug development. The results of our earlier work suggested that hydrophobic statins induce autophagy in cultured human rhabdomyosarcoma A204 cells. In this study, we first confirmed the statin-induced autophagy by assessing other criteria, including induced expression of the autophagy-related genes, enhanced protein degradation of autophagy marker protein p62 and electron microscopic observation of induced formation of autophagosome. We next demonstrated that the extent of inhibition of HMG-CoA reductase in the cell is parallel with the ability of a statin to induce autophagy. Thus, the primary activity of statins causes autophagy in A204 cells. Considering the mechanism for the induction, we showed that statins induce autophagy by depleting cellular levels of geranylgeranyl diphosphate (GGPP) mostly through an unknown pathway that does not involve two major small G proteins, Rheb and Ras. Finally, we demonstrated that the ability of statins to induce autophagy parallels their toxicity to A204 cells and that both can be suppressed by GGPP.

Angiotensin II-dependent hypertension causes reversible changes in the platelet proteome

Hypertension is a risk factor for arterial thrombosis. We investigated the effects of angiotensin II (ANG II)-dependent hypertension on the platelet proteome. Hypertension was induced in cyp1a1ren-2 transgenic rats by feeding indole-3-carbinol (n = 10) and in Fischer 344 rats by subcutaneously infusing ANG II (n = 7). After 14 days of hypertension (SBP 180 mmHg) and 10 days after normalization of blood pressure, changes in the platelet proteome were assessed by two-dimensional differential in-gel electrophoresis. In a subset of animals (n = 4), repeated blood withdrawals were performed. Of 1040 protein spots, 45 displayed hypertension-associated changes (>1.5-fold, P < 0.01) in both models (34 increased, 11 decreased). All were reversible within 10 days. Thirty-eight spots were identified by mass spectrometry and assigned to 20 distinct proteins. The majority of spots with increased intensity constituted protein fragments. Repeated blood withdrawals and stimulation of megakaryocytopoiesis by a thrombopoietin receptor agonist induced changes in the same protein spots but in the opposite direction to those induced by ANG II-dependent hypertension. ANG II-dependent hypertension is associated with enhanced protein degradation in platelets. As these changes are reversible, the proteins identified might be used to develop a biomarker for monitoring recent blood pressure history.

AMP-Activated Protein Kinase Regulates E3 Ligases in Rodent Heart

The degradation of proteins by the ubiquitin proteasome system (UPS) is required for the maintenance of cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). AMPK activation inhibits protein synthesis and activates autophagy, but whether AMPK plays a role in regulating protein breakdown through the UPS in the heart is not known. To determine whether AMPK enhances UPS-mediated protein degradation by directly regulating the ubiquitin ligases Atrogin-1 and muscle RING finger protein 1 (MuRF1) in the heart. Nutrient deprivation and pharmacological or genetic activation of AMPK increased mRNA expression and protein levels of Atrogin-1 and MuRF1 and consequently enhanced protein degradation in neonatal cardiomyocytes. Inhibition of AMPK abrogated these effects. Using gene reporter and chromatin immunoprecipitation assays, we found that AMPK regulates MuRF1 expression by acting through the myocyte enhancer factor 2 (MEF2). We further validated these findings in vivo using MEF2-LacZ reporter mice. Furthermore, we demonstrated in adult cardiomyocytes that MuRF1 is necessary for AMPK-mediated proteolysis through the UPS in the heart. Consequently, MuRF1 knockout mice were protected from severe cardiac dysfunction during fasting. AMPK regulates the transcription of Atrogin-1 and MuRF1 and enhances UPS-mediated protein degradation in heart. Specifically, AMPK regulates MuRF1 through the transcription factor MEF2. The absence of MuRF1 in the heart preserves cardiac function during fasting. The results strengthen the hypothesis that AMPK serves as a modulator of intracellular protein degradation in the heart.

Trimming of Ubiquitin Chains by Proteasome-associated Deubiquitinating Enzymes

The proteasome generally recognizes substrate via its multiubiquitin chain followed by ATP-dependent unfolding and translocation of the substrate from the regulatory particle into the proteolytic core particle to be degraded. Substrate-bound ubiquitin groups are for the most part not delivered to the core particle and broken down together with substrate but instead recovered as intact free ubiquitin and ubiquitin chains. Substrate deubiquitination on the proteasome is mediated by three distinct deubiquitinating enzymes associated with the regulatory particle: RPN11, UCH37, and USP14. RPN11 cleaves at the base of the ubiquitin chain where it is linked to the substrate, whereas UCH37 and apparently USP14 mediate a stepwise removal of ubiquitin from the substrate by disassembling the chain from its distal tip. In contrast to UCH37 and USP14, RPN11 shows degradation-coupled activity; RPN11-mediated deubiquitination is apparently delayed until the proteasome is committed to degrade the substrate. Accordingly, RPN11-mediated deubiquitination promotes substrate degradation. In contrast, removal of ubiquitin prior to commitment could antagonize substrate degradation by promoting substrate dissociation from the proteasome. Emerging evidence suggests that USP14 and UCH37 can both suppress substrate degradation in this way. One line of study has shown that small molecule USP14 inhibitors can enhance proteasome function in cells, which is consistent with this model. Enhancing protein degradation could potentially have therapeutic applications for diseases involving toxic proteins that are proteasome substrates. However, the responsiveness of substrates to inhibition of proteasomal deubiquitinating enzymes may vary substantially. This substrate specificity and its mechanistic basis should be addressed in future studies.

Skeletal Muscle Oxidative Capacity in Cachectic Mice

Cachexia is often defined as a complex metabolic syndrome associated with underlying illness and characterized by the loss of muscle with or without loss of fat mass. These changes are often attributed to chronic inflammation that can enhance protein degradation in skeletal muscle and lypolysis in adipose tissue. The purpose of this study was to examine if the enhanced metabolic demands during cachexia alter oxidative capacity in red and white portions of the gastrocnemius muscle from cachectic ApcMin/+ mice. Twenty week ApcMin/+ mice were stratified by cachexia severity determined by body weight, muscle, and fat mass. The gastrocnemius muscle was separated into red and white portions to determine phenotype specific changes. Muscle mitochondrial content and succinate dehydrogenase activity were significantly reduced in mice with severe cachexia. Gene expression related to mitochondrial fission and fusion were quantified in the muscle. In glycolytic muscle there was a significant decrease in mfn1 and mfn2 expression, and an increase in fis1 mRNA expression. Nitrotyrosine expression, a marker of oxidative stress was significantly decreased in both oxidative and glycolytic muscle from severely cachectic mice. These results suggest that during severe cachexia skeletal muscle mitochondria number and function are altered in red and white portions of rapidly wasting skeletal muscle. RO1CA121249‐01A2

Relationship between N-acetyltransferase 2 single-nucleotide polymorphisms and aromatic DNA adducts

Relationship between N-acetyltransferase 2 single-nucleotide polymorphisms and aromatic DNA adducts

Defects in chromosome congression and mitotic progression in KIF18A-deficient cells are partly mediated through impaired functions of CENP-E

ABSTRACTKIF18A, a molecular motor, is an essential component in the regulation of orderly chromosome congression by attenuation of the kinetochore oscillation amplitude at the midzone during mitosis in vertebrate cells. Here we report that KIF18A depletion resulted in mitotic arrest which was accompanied by the presence of unaligned chromosomes in HeLa cells. This resembles the phenotype induced by an impaired function of CENP-E, also a mitotic kinesin essential for the formation of the mitotic spindles. Our further analysis showed that KIF18A depletion caused specific down-regulation of CENP-E. Down-regulation of CENP-E as the result of KIF18A silencing was not due to reduced transcription but primarily due to the enhanced protein degradation. Co-immunoprecipitation revealed that KIF18A physically interacted with CENP-E and BubR1 during mitosis. Ectopic expression of the wild-type tail domain of CENP-E, but not a corresponding mutant, significantly suppressed chromosome congression defects in mitotic cells. Together, our studies strongly suggest that chromosome congression defects as the result of KIF18A depletion is at least in part mediated through destabilizing kinetochore CENP-E.

AIP-1 ameliorates β-amyloid peptide toxicity in a Caenorhabditis elegans Alzheimer's disease model

Multiple neurodegenerative diseases are causally linked to aggregation-prone proteins. Cellular mechanisms involving protein turnover may be key defense mechanisms against aggregating protein disorders. We have used a transgenic Caenorhabditis elegans Alzheimer's disease model to identify cellular responses to proteotoxicity resulting from expression of the human beta amyloid peptide (Abeta). We show up-regulation of aip-1 in Abeta-expressing animals. Mammalian homologues of AIP-1 have been shown to associate with, and regulate the function of, the 26S proteasome, leading us to hypothesize that induction of AIP-1 may be a protective cellular response directed toward modulating proteasomal function in response to toxic protein aggregation. Using our transgenic model, we show that overexpression of AIP-1 protected against, while RNAi knockdown of AIP-1 exacerbated, Abeta toxicity. AIP-1 overexpression also reduced accumulation of Abeta in this model, which is consistent with AIP-1 enhancing protein degradation. Transgenic expression of one of the two human aip-1 homologues (AIRAPL), but not the other (AIRAP), suppressed Abeta toxicity in C. elegans, which advocates the biological relevance of the data to human biology. Interestingly, AIRAPL and AIP-1 contain a predicted farnesylation site, which is absent from AIRAP. This farnesylation site was shown by others to be essential for an AIP-1 prolongevity function. Consistent with this, we show that an AIP-1 mutant lacking the predicted farnesylation site failed to protect against Abeta toxicity. Our results implicate AIP-1 in the regulation of protein turnover and protection against Abeta toxicity and point at AIRAPL as the functional mammalian homologue of AIP-1.

Growth Factor Withdrawal: A Novel Approach To In Vitro Muscle Atrophy

PURPOSE: Skeletal muscle disuse is associated with decreases in muscle size and function resulting from reduced protein synthesis and enhanced protein degradation. Current in vitro models of atrophy may not adequately approximate in vivo disuse because the atrophic phenotype is elicited through potent cytokine or glucocorticoid treatment. A cell culture model using methods that are more physiologically analogous to disuse, such as the modulation of growth factor availability, may provide a useful tool to study the molecular mechanisms associated with disuse atrophy. Therefore, the purpose of this study was to characterize a novel means to induce atrophy using growth factor withdrawal of C2C12 myotubes. METHODS: C2C12 myoblasts were incubated in medium (DMEM) supplemented with 10% fetal bovine serum, a growth factor rich medium (GM). At 80-90% confluence, cells were transferred to DMEM supplemented with 2% horse serum, a relatively growth factor deficient differentiation medium (DM) to stimulate myotube formation. Following 4 days in DM, fused myotubes were re-introduced into GM to induce hypertrophy. After 48 hours, the GM was replaced again with DM to induce atrophy. Immunocytochemistry (ICC), fluorescence microscopy, and Metavue analytical software package were used to visualize and quantify myotube diameter at baseline, 24, 48 and 72 hours after placing the myotubes in DM. Western blotting was used to determine levels of MHC protein and qRT-PCR was used to measure expression of the ubiquitin E3 ligases atrogin1 and MuRF1. RESULTS: Myotubes had a significant 39 and 41% decrease in myotube diameter following 48 and 72 hours of growth factor withdrawal respectively. Western blotting revealed a significant decrease in MHC following 24 and 72 hours of growth factor withdrawal. Atrogin1 mRNA expression was unchanged and MuRF1 mRNA was significantly upregulated by 2.9 and 2.5 fold at 24 and 48 hours post-growth factor withdrawal, respectively. CONCLUSION: These data indicate that growth factor withdrawal produces an atrophy response in C2C12 myotubes. Furthermore, because the atrophic phenotype does not require the treatment of cultures with cytokines or glucocorticoids, but rather is a simple manipulation of standard cell culture media, it may provide advantages over currently used models of atrophy.

NEDD4 mediated degradation of target proteins under simulated microgravity conditions

The effects of microgravity on human vascular endothelial cells may contribute to altered cardiovascular functions in space. Since endothelial function critically depends on protein synthesis and degradation, we addressed the effects of microgravity on the ubiquitin‐ligase NEDD4 in endothelial cells. Human umbilical vein endothelial cells or EAhy926 cells were exposed to simulated microgravity in the Rotating Wall Vessel (RWV) for 24 h, 48 h and 72 h. Controls were kept under normal gravity or exposed to shear stress using a cone and plate apparatus (6 dyn\cm2, 24 h). Gene expression and protein concentration were analysed by real‐time RT‐PCR and Western blotting, respectively. Our results show a two fold upregulation of NEDD4 under microgravity and no regulation by shear stress (relative to controls 1G, no shear). NEDD4 targets e.g. Cx43, Notch1, eps15, IGF1R, PLCgamma1 were strongly downregulated after 48 h and 72 h exposure to microgravity. These data suggest that NEDD4 expression is sensitive to gravity, which may be a mechanism to enhance protein degradation in endothelial cells.

IL-6 and Serum Amyloid A Synergy Mediates Angiotensin II–Induced Muscle Wasting

Animal studies suggest that increased levels of circulating angiotensin II (AngII) could contribute to the loss of lean body mass in chronic kidney disease, but the mechanism by which this occurs is unclear. Here, AngII infusion increased circulating IL-6 and its hepatic production in wild-type mice, suggesting that AngII-induced inflammation may trigger muscle loss. AngII infusion also stimulated the suppressor of cytokine signaling (SOCS3) in muscle, which led to loss of insulin receptor substrate 1 (IRS-1), thereby impairing insulin/IGF-1 signaling and enhancing protein degradation. All of these responses to AngII were suppressed in IL-6-deficient mice. Recombinant human IL-6 (rhIL-6) treatment of cultured myotubes only minimally increased SOCS3, however, suggesting the contribution of other mediators. Because AngII increases hepatic serum amyloid A (SAA) expression in an IL-6-dependent manner, we treated wild-type mice with rhIL-6 and an SAA1-overexpressing adenovirus; the combination led to a significantly greater increase in SOCS3 and decrease in IRS-1 compared with either rhIL-6 or SAA1 alone. We observed similar effects on SOCS3 and IRS-1 when we treated cultured muscle myotubes with rhIL-6 and SAA1. Taken together, these results suggest an interorgan response to high levels of AngII: Hepatic production of IL-6 and SAA increases, and these mediators act synergistically to impair insulin/IGF-1 signaling, which promotes muscle proteolysis. Targeting the high levels of IL-6 and SAA in catabolic disorders might be a therapeutic approach to prevent muscle wasting.

Resistin increases lipid accumulation by affecting class A scavenger receptor, CD36 and ATP-binding cassette transporter-A1 in macrophages

Aims Resistin promotes macrophage-foam cell formation, but the mechanisms are unclear. In macrophages, lipid uptake is regulated by scavenger receptors (SR-A and CD36), while the cholesterol efflux is regulated by SR-BI, ATP-binding cassette transporter-A1 (ABCA1) and ABCG1. We investigated the mechanisms underlying the dysregulation by resistin of these regulators leading to promotion of lipid accumulation in bone marrow-derived macrophages. Main methods Western blotting, real-time PCR and oil red O staining were performed. Key findings Resistin exacerbated lipid accumulation in oxLDL-treated macrophages. Resistin treatment of oxLDL-untreated macrophages showed increased SR-A and CD36 mRNA and protein levels, and decreased ABCA1 protein level, while having no effect on SR-BI or ABCG1 expression. Up-regulation of SR-A and CD36 by resistin resulted from activation of AP-1 and PPARγ, respectively, and this was confirmed by the lack of activation of either after AP-1 inhibition using curcumin or SP600125, or PPARγ inhibition using GW9662, respectively. The down-regulation of ABCA1 by resistin was not accompanied by a reduced mRNA level or an activation of LXRα/RXR, but resulted from enhanced protein degradation as revealed by the abolition of the down-regulation after inhibition of the proteasome pathway using ALLN or MG-132. A combined inhibition by SP600125, GW9662 and ALLN prevented resistin-induced exacerbation of lipid accumulation in oxLDL-treated macrophages. Significance Resistin promotes foam cell formation via dysregulation of SR-A, CD36 and ABCA1. SR-A and CD36 are transcriptionally up-regulated by resistin through AP-1 and PPARγ, respectively, whereas ABCA1 is down-regulated by resistin through proteasome-mediated enhancement of protein degradation.

Therapeutic application of histone deacetylase inhibitors for central nervous system disorders

Histone deacetylases (HDACs)--enzymes that affect the acetylation status of histones and other important cellular proteins--have been recognized as potentially useful therapeutic targets for a broad range of human disorders. Pharmacological manipulations using small-molecule HDAC inhibitors--which may restore transcriptional balance to neurons, modulate cytoskeletal function, affect immune responses and enhance protein degradation pathways--have been beneficial in various experimental models of brain diseases. Although mounting data predict a therapeutic benefit for HDAC-based therapy, drug discovery and development of clinical candidates face significant challenges. Here, we summarize the current state of development of HDAC therapeutics and their application for the treatment of human brain disorders such as Rubinstein-Taybi syndrome, Rett syndrome, Friedreich's ataxia, Huntington's disease and multiple sclerosis.

Can herbage nitrogen fractionation in Lolium perenne be improved by herbage management?

The high degradability of grass protein is an important factor in the low nitrogen (N) utilization of grazing bovines in intensive European grassland systems. We tested the hypothesis that protein degradability as measured by the Cornell Net Carbohydrate and Protein System (CNCPS) protein fractionation scheme, can be manipulated by herbage management tools, with the aim to reduce N loss to the environment. A field experiment comprising the factorial combinations of three fertilizer N application rates (0, 90 and 390 kg N ha −1 year −1), three regrowth periods (2–3, 4–5, and 6–7 weeks), two perennial ryegrass ( Lolium perenne L.) cultivars [Aberdart (high sugar content) and Respect (low sugar content)] and two cutting heights (approximately 8 and 12 cm) was conducted at Teagasc, Johnstown Castle Research Centre, Wexford, Ireland. The plots were sampled during four seasons [September/October 2002 (late season), April 2003 (early season), May/June 2003 (mid season) and September 2003 (late season)] and protein fractions were determined in both sheath and lamina material. The protein was highly soluble and on average 19% and 28% of total N was in the form of non-protein N, 16% and 19% in the form of buffer-soluble protein, 52% and 40% in the form of buffer-insoluble protein, and 12% and 13% in the form of potentially available cell wall N for lamina and sheath material, respectively. In both materials only 0.9% of total N was present as unavailable cell wall N. In general the herbage management tools investigated did not have much effect on protein fractionation. The effects of regrowth period, cultivar and cutting height were small and inconsistent. High N application rates significantly increased protein degradability, especially during late season. This is relevant, as it has been shown that enhanced protein degradation increases the potential N loss through urine excretion at a time when urine-N excreted onto pasture is prone to leaching. However, the effect was most evident for sheath material, which forms only a small proportion of the animals' intake. It was concluded that there appears to be little scope for manipulating the herbage-N fractionation through herbage management. The consequences for modelling herbage quality could be positive as there does not seem to be a need to model the individual N fractions; in most cases the N fractions can be expressed as a fixed proportion of total N instead.

Induction of NEDD4 under simulated microgravity

Vascular endothelial cells are exposed to mechanical forces e.g. wall shear stress, wall tension, and pulsatile stretch, which are modulated by the gravitational field. The effects of microgravity may influence endothelial cell function including protein synthesis and degradation.Therefore, this study was designed to investigate microgravity dependent effects on the ubiquitin‐ligase NEDD4 in endothelial cells. In order to produce microgravity endothelial cells (EA hy926, HUVEC) were seeded on microcarriers and cultured in the Rotating Wall Vessel for 24 h and 48 h. Controls were kept under normal gravity or exposed to shear stress using a cone and plate apparatus (6 dyn\cm2, 24 h). Gene expression and protein concentration were analysed by real‐time RT‐PCR and Western blotting, respectively. Our results indicate that NEDD4 is upregulated to 200% under microgravity and downregulated to 70% by shear stress relative to controls (1G, no shear). The present data suggest that NEDD4 is sensitive to mechanical forces; the induction under microgravity may be a mechanism to enhance protein degradation in endothelial cells.

Caveolin‐1 facilitates cyclooxygenase‐2 degradation through a p97/valosin‐containing protein dependent pathway

Cyclooxygenase (COX)‐2 participates in various physiological functions and involves in tumorigenesis. Although transactivation of COX‐2 has been well studied, the factor that responsible for COX‐2 degradation remains mostly unknown. In this study, COX‐2 degradation induced by caveolin‐1 (Cav‐1) is demonstrated by upregulated endogenous and bacteria induced COX‐2 in lung and colon of caveolin‐1 null mice. Ectopic expression of caveolin‐1 in HT‐29 cells resulted in suppressed endogenous and IL‐1β induced COX‐2 expression. Cav‐1‐induced COX‐2 degradation was implicated to enhanced protein degradation by the ubiquitin‐proteosome pathway. Via immunoprecipitation, we identified valosin‐containing protein (VCP/p97) as the chaperon protein which interacts with both Cav‐1 and COX‐2 and specifically mediates COX‐2, but not COX‐1, degradation. Suppression of VCP via siRNA resulted in increased COX‐2 level with prolonged half‐life. Moreover, deletion of the COX‐2 C‐terminal 32 amino acids upregulated its expression level and lengthened its half‐life. These data reveal a novel degradation mechanism that Cav‐1 and VCP synergistically orchestrate COX‐2 degradation, suggesting a role of Cav‐1 in anti‐tumorigenesis.

Influences of lipopolysaccharide-induced immune challenge on performance and whole-body protein turnover in weanling pigs

The objective of this study is to characterize the influence of immune stress induced by lipopolysaccharide (LPS) on protein utilization and turnover for early-weaned pigs. A total of 15, crossbred weanling pigs (initial body weight 10.15 ± 0.39 kg) were assigned to one of three treatments. Pigs were injected with LPS and fed ad libitum (LPS-challenge), or injected with endotoxin-free physiological salt solution (PSS) and fed ad libitum (control), or injected with PSS and fed the same amount of feed as LPS-challenged pigs (pair-feed). All pigs received a 4-d nitrogen balance trial. On d 1 and 3 of the trial, LPS-challenged pigs were injected intramuscularly with 200 μg/kg BW of LPS dissolved in 1 ml PSS. Pigs in other treatments were injected with 1 ml PSS. 15 N-Glycine (5 mg/kg BW) was gastrically infused after the second injection. Feces and urine were collected daily to determine the N output throughout the duration of the trial. Lymphocyte blastogenesis (LB) and serum immunoglobulins (IgA, IgG, IgM) were also detected to illustrate the LPS-induced immune responses. Results indicated that the injection of LPS significantly ( P < 0.01) elevated the LB and resulted in lower average daily gain (ADG), feed intake (ADFI) and efficiency of feed utilization than control pigs. Pair-fed pigs had higher performance than LPS-challenged pig but poorer than control pigs. Injection of LPS also resulted in significantly ( P < 0.01) lower nitrogen intake and efficiency of utilization than controls, and more fecal N excretion than Pair-fed pigs (3.19 ± 0.85 vs. 2.19 ± 0.67 g/d, P < 0.05). The whole-body nitrogen flux (4.34 ± 0.19 vs. 11.35 ± 0.12 g N/kg BW 0.75/d) and N accretion (5.57 ± 0.59 vs. 10.17 ± 1.12 g Pr/kg BW 0.75/d) were acutely ( P < 0.01) reduced as the feed intake decreased, but there was no significant difference between LPS-challenged and pair-fed pigs. Injection of LPS markedly ( P < 0.05) increased the protein degradation (16.76 ± 1.09 vs. 14.53 ± 1.24 g N/kg BW 0.75/d). It is concluded that LPS-induced immune challenge depresses growth performance and feed utilization efficiency by enhancing protein degradation rate and decreasing protein utilization for body protein retention.

Overexpression of the S-phase kinase-associated protein 2 in thyroid cancer

Loss of expression of the cyclin-dependent kinase inhibitor p27 through enhanced protein degradation frequently occurs in human cancer. Degradation of p27 requires ubiquitination by the S-phase kinase-associated protein 2 (Skp2), a member of the F-box family of Skp1-Cullin-F-box protein ubiquitin ligases. In the present study, we have investigated the role of Skp2 in human thyroid tumours. Immunohistochemistry analysis showed that Skp2 was overexpressed significantly in thyroid carcinomas (26 out of 51) compared with goitres (0 out of 12, P<0.001) or adenomas (1 out of 10, P<0.05), and that high Skp2 expression was detected more often in anaplastic thyroid (ATC; 83%, n=12) than follicular thyroid (FTC; 40%, n=20) or papillary thyroid (PTC; 42%, n=19) carcinomas (P<0.05). Thyroid cancer cell lines and tissues with high levels of Skp2 protein presented high p27 degradation activity and there was an inverse correlation between Skp2 and p27 expression in thyroid cancer tissues (n=68; P<0.05). In most cases, the observed overexpression of Skp2 protein was paralleled by an increase in the levels of Skp2 mRNA, and we observed Skp2 gene amplification at 5p13 in 2 out of 6 cell lines and in 9 out of 23 primary tumours (six out of eight ATCs, two out of nine PTCs and one out of six FTCs) using Q-PCR and/or fluorescence in situ hybridization analysis. Finally, in vitro experiments demonstrated that suppression of Skp2 expression drastically reduced proliferation of thyroid cancer cells and, conversely, forced expression of Skp2 circumvented serum dependency and contact inhibition in Skp2-negative cells by promoting p27 degradation. These findings indicate that Skp2 plays an important role for the development of thyroid cancer.

Cell Biology Assessment of Glucokinase Mutations V62M and G72R in Pancreatic β-Cells

Mutations in the glucokinase (GK) gene cause defects in blood glucose homeostasis. In some cases (V62M and G72R), the phenotype cannot be explained by altered enzyme kinetics or protein instability. We used transient and stable expression of green fluorescent protein (GFP) GK chimaeras in MIN6 beta-cells to study the phenotype defect of V62M and G72R. GK activity in lysates of MIN6 cell lines stably expressing wild-type or mutant GFP GK showed the expected affinity for glucose and response to pharmacological activators, indicating the expression of catalytically active enzymes. MIN6 cells stably expressing GFP V62M or GFP G72R had a lower GK activity-to-GK immunoreactivity ratio and GK activity-to-GK mRNA ratio but not GK immunoreactivity-to-GK mRNA ratio than wild-type GFP GK. Heterologous expression of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/FDP2) in cell lines increased GK activity for wild-type GK and V62M but not for G72R, whereas expression of liver GK regulatory protein (GKRP) increased GK activity for wild type but not V62M or G72R. Lack of interaction of these mutants with GKRP was also evident in hepatocyte transfections from the lack of nuclear accumulation. These results suggest that cellular loss of GK catalytic activity rather than impaired translation or enhanced protein degradation may account for the hyperglycemia in subjects with V62M and G72R mutations.

Posttranscriptional Inhibition of Interferon-Gamma Production by Lead

Lead (Pb) is known to preferentially suppress the activation and development of type-1 CD4+ helper T cell (Th1) responses, whereas it enhances the development of type-2 CD4+ helper T cell (Th2) responses. The inhibition of interferon-gamma (IFNgamma) production has been demonstrated in vitro with a Th1 clone and DO11.10 ovalbumin-transgenic (OVA-tg) CD4+ T cells, and in vivo with wild-type and OVA-tg BALB/c mice; however, the mechanisms responsible for the Pb-induced downregulation of IFNgamma have not been reported. Here, we assessed the modulation of IFNgamma production at the mRNA and protein levels. Pb did not significantly affect IFNgamma mRNA expression by a Th1 clone or activated splenocytes, as measured by reverse transcriptase-polymerase chain reaction (RT-PCR), ribonuclease protection, and real-time RT-PCR. However, Pb did significantly lower the amount of IFNgamma protein in supernatants and cell lysates of antigen-activated T cells in comparison to stimulated controls, suggesting that the lower amounts of IFNgamma released into culture supernatants were not due to a blockage of secretion that gave rise to a cytoplasmic accumulation of IFNgamma. Pb inhibition also was not prevented by addition of zinc or iron. Pb did not enhance protein degradation of IFNgamma, in that lactacystin, an effective blocker of proteosomal proteolysis, did not prevent loss of IFNgamma; additionally, Pb did not accelerate loss of IFNgamma after cycloheximide treatment. Pb did, however, significantly suppress IFNgamma biosynthesis, as investigated using 35S-incorporation in pulse/chase experiments, although it did not suppress total protein synthesis, indicating that Pb selectively inhibits IFNgamma biosynthesis. Thus, Pb appears to selectively interfere with the translation of certain proteins, such as IFNgamma. IL-12 blocked Pb's preferential promotion of Th2 cells, but absence of STAT6 did not prevent the Pb skewing. Thus, Pb may modulate unique regulatory pathways.