Neil J. Shimwell

Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells

Mammalian cells contain different phospholipase D enzymes (PLDs) whose distinct physiological roles are poorly understood and whose products have not been characterized. The development of porcine aortic endothelial (PAE) cell lines able to overexpress PLD-1b or -2a under the control of an inducible promoter has enabled us to characterize both the substrate specificity and the phosphatidic acid (PtdOH) product of these enzymes under controlled conditions. Liquid chromatography-MS analysis showed that PLD1b- and PLD2a-transfected PAE cells, as well as COS7 and Rat1 cells, generate similar PtdOH and, in the presence of butan-1-ol, phosphatidylbutanol (PtdBut) profiles, enriched in mono- and di-unsaturated species, in particular 16:0/18:1. Although PtdBut mass increased, the species profile did not change in cells stimulated with ATP or PMA. Overexpression of PLD made little difference to basal or stimulated PtdBut formation, indicating that activity is tightly regulated in vivo and that factors other than just PLD protein levels limit hydrolytic function. In vitro assays using PLD-enriched lysates showed that the enzyme could utilize both phosphatidylcholine and, much less efficiently, phosphatidylethanolamine, with slight selectivity towards mono- and di-unsaturated species. Phosphatidylinositol was not a substrate. Thus PLD1b and PLD2a hydrolyse a structurally similar substrate pool to generate an identical PtdOH product enriched in mono- and di-unsaturated species that we propose to function as the intracellular messenger forms of this lipid.

Adipsin and the glucose transporter GLUT4 traffic to the cell surface via independent pathways in adipocytes.

Insulin increases the exocytosis of many soluble and membrane proteins in adipocytes. This may reflect a general effect of insulin on protein export from the trans Golgi network. To test this hypothesis, we have compared the trafficking of the secreted serine protease adipsin and the integral membrane proteins GLUT4 and transferrin receptors in 3T3‐L1 adipocytes. We show that adipsin is secreted from the trans Golgi network to the endosomal system, as ablation of endosomes using transferrin‐HRP conjugates strongly inhibited adipsin secretion. Phospholipase D has been implicated in export from the trans Golgi network, and we show that insulin stimulates phospholipase D activity in these cells. Inhibition of phospholipase D action with butan‐1‐ol blocked adipsin secretion and resulted in accumulation of adipsin in trans Golgi network‐derived vesicles. In contrast, butan‐1‐ol did not affect the insulin‐stimulated movement of transferrin receptors to the plasma membrane, whereas this was abrogated following endosome ablation. GLUT4 trafficking to the cell surface does not utilise this pathway, as insulin‐stimulated GLUT4 translocation is still observed after endosome ablation or inhibition of phospholipase D activity. Immunolabelling revealed that adipsin and GLUT4 are predominantly localised to distinct intracellular compartments. These data suggest that insulin stimulates the activity of the constitutive secretory pathway in adipocytes possibly by increasing the budding step at the TGN by a phospholipase D‐dependent mechanism. This may have relevance for the secretion of other soluble molecules from these cells. This is not the pathway employed to deliver GLUT4 to the plasma membrane, arguing that insulin stimulates multiple pathways to the cell surface in adipocytes.

Combined proteome and transcriptome analyses for the discovery of urinary biomarkers for urothelial carcinoma

Background:Proteomic discovery of cancer biomarkers in body fluids is challenging because of their low abundance in a complex background. Altered gene expression in tumours may not reflect protein levels in body fluids. We have tested combining gene expression profiling of tumours with proteomic analysis of cancer cell line secretomes as a strategy to discover urinary biomarkers for bladder cancer.Methods:We used shotgun proteomics to identify proteins secreted by three bladder cancer cell lines. Secreted proteins with high mRNA levels in bladder tumours relative to normal urothelium were assayed by ELISA in urine samples from 642 patients.Results:Midkine and HAI-1 were significantly increased in bladder cancer patients, with the highest levels in invasive disease (area under the receiver operating characteristic curve 0.89 vs non-cancer). The urinary concentration of both proteins was too high to be explained by bladder cancer associated haematuria and most likely arises by direct tumour secretion.Conclusions:This ‘dual-omic’ strategy identified tumour secreted proteins whose urine concentrations are increased significantly by bladder cancer. Combined secretome-transcriptome analysis may be more useful than direct proteomic analysis of body fluids for biomarker discovery in both bladder cancer and other tumour types.

Assessment of high-throughput high-resolution MALDI-TOF-MS of urinary peptides for the detection of muscle-invasive bladder cancer.

Purpose: There is a need for better biomarkers to both detect bladder cancer and distinguish muscle‐invasive (stage T2+) from non‐invasive (stage Ta/T1) disease. We assess whether MALDI‐TOF‐MS of the urine peptidome can achieve this.

Urinary EpCAM in urothelial bladder cancer patients: characterisation and evaluation of biomarker potential

Background:Epithelial cell adhesion molecule is overexpressed in bladder tumours and released from bladder cancer cells in vitro. We test the hypotheses that urinary EpCAM could act as a biomarker for primary bladder cancer detection and risk stratification.Methods:Epithelial cell adhesion molecule was measured by ELISA in urine from 607 patients with primary bladder tumours and in urine from 53 non-cancer controls. Mann–Whitney tests and ROC analyses were used to determine statistical significance and discrimination between non-cancer controls and different stages and grades of disease. Multivariable modelling and Kaplan–Meier analyses were used to determine prognostic significance. The structure of urinary EpCAM was investigated by western blotting and mass spectrometry.Results:Urinary EpCAM levels increase with stage and grade of bladder cancer. Alongside grade and stage, elevated urinary EpCAM is an independent indicator of poor prognosis with a hazard ratio of 1.76 for bladder cancer-specific mortality. The soluble form of EpCAM in urine is the extracellular domain generated by cleavage between ala243 and gly244. Further studies are required to define the influence of other urinary tract malignancies and benign urological conditions on urinary EpCAM.Conclusion:The extracellular domain of EpCAM is shed into urine by bladder tumours. Urinary EpCAM is a strong indicator of bladder cancer-specific survival, and may be useful within a multi-marker panel for disease detection or as a stand-alone marker to prioritise the investigation and treatment of patients. The mechanisms and effects of EpCAM cleavage in bladder cancer are worthy of further investigation, and may identify novel therapeutic targets.

Adenovirus E4orf3 Targets Transcriptional Intermediary Factor 1γ for Proteasome-Dependent Degradation during Infection

ABSTRACT The ability of adenovirus early region proteins, E1B-55K and E4orf6, to usurp control of cellular ubiquitin ligases and target proteins for proteasome-dependent degradation during infection is well established. Here we show that the E4 gene product, E4orf3 can, independently of E1B-55K and E4orf6, target the transcriptional corepressor transcriptional intermediary factor 1γ (TIF1γ) for proteasome-mediated degradation during infection. Initial mass spectrometric studies identified TIF1 family members—TIF1α, TIF1β, and TIF1γ—as E1B-55K-binding proteins in both transformed and infected cells, but analyses revealed that, akin to TIF1α, TIF1γ is reorganized in an E4orf3-dependent manner to promyelocytic leukemia protein-containing nuclear tracks during infection. The use of a number of different adenovirus early region mutants identified the specific and sole requirement for E4orf3 in mediating TIF1γ degradation. Further analyses revealed that TIF1γ is targeted for degradation by a number of divergent human adenoviruses, suggesting that the ability of E4orf3 to regulate TIF1γ expression is evolutionarily conserved. We also determined that E4orf3 does not utilize the Cullin-based ubiquitin ligases, CRL2 and CRL5, or the TIF1α ubiquitin ligase in order to promote TIF1γ degradation. Further studies suggested that TIF1γ possesses antiviral activity and limits adenovirus early and late gene product expression during infection. Indeed, TIF1γ knockdown accelerates the adenovirus-mediated degradation of MRE11, while TIF1γ overexpression delays the adenovirus-mediated degradation of MRE11. Taken together, these studies have identified novel adenovirus targets and have established a new role for the E4orf3 protein during infection.

Assessment of novel combinations of biomarkers for the detection of colorectal cancer

BACKGROUND Patients with colorectal cancer often present with advanced disease and concomitant poor prognosis. The best known serum biomarker, carcinoembryonic antigen (CEA) is not recommended for screening because of its limited specificity and sensitivity. A number of other circulating proteins have been suggested to be diagnostically useful but individually none of these has proved to be of sufficient sensitivity or specificity to establish a role in routine clinical practice. Here, we test the hypothesis that combining several of these biomarkers will improve diagnostic efficacy. METHODS To select the markers for our model we screened CEA and 26 other candidate biomarkers. Four candidates were selected and their concentrations determined in the serum of 239 patients (106 colorectal cancer patients and 133 non-cancer subjects). RESULTS Class prediction models based on CEA, DR-70 and sCD26 produced a modest increase in detection accuracy over CEA alone, particularly for early stage cancers. The sensitivity and specificity required for a clinically useful test was not reached. CONCLUSION It is unlikely that a biomarker panel comprised of the currently available serum markers will generate a clinically useful diagnostic test for colorectal cancer. Our findings reiterate the urgent need to discover novel biomarkers for the detection of colorectal cancer.

Adenovirus 5 E1A is responsible for increased expression of insulin receptor substrate 4 in established adenovirus 5-transformed cell lines and interacts with IRS components activating the PI3 kinase/Akt signalling pathway

Using mass spectrometric analysis insulin receptor substrate 4 (IRS-4) has been identified as a novel adenovirus 5 early region 1A (Ad5E1A)-binding protein. IRS-4 interacts with both the transcriptional activation domain (conserved region 3) and the N-terminal region of Ad5E1A13S. Prolonged expression of Ad5E1A13S is required for the observed dramatic increase in the levels of IRS-4 mRNA and protein in Ad5E1-transformed human cell lines. Once expressed, as well as binding to E1A and the insulin receptor, IRS-4 remains tyrosine phosphorylated and constitutively associates with the regulatory p85 subunit of phosphoinositide 3 kinase, resulting in the phosphorylation of Akt (causing activation) and GSK-3β (causing inhibition). Reducing IRS-4 expression using small interfering RNA (siRNA) in established Ad5E1A-expressing cell lines decreases the activation of Akt and cellular proliferation. During Ad5 infection, IRS-4 is not expressed. However, Ad5E1A associates with IRS-1, increasing Akt and GSK-3β phosphorylation and tyrosine phosphorylation of IRS-1 itself. We conclude that the association and altered regulation of IRS proteins by Ad5E1A contribute to the adenovirus-transformed phenotype and modulates viral infection in an Akt-dependent manner.

Proteomic profiling of N-linked glycoproteins identifies ConA-binding procathepsin D as a novel serum biomarker for hepatocellular carcinoma

The aim of this study was to identify novel biomarkers for the diagnosis of, and potential therapeutic targets for, hepatocellular carcinoma (HCC). Multilectin affinity chromatography was used to enrich N‐linked glycoproteins from nontumorous liver and HCC tissues followed by 2DE and protein identification by MS. Twenty‐eight differentially expressed proteins were identified. Western blotting validated consistently lower concentrations of human liver carboxylesterase 1 and haptoglobin, and higher concentration of procathepsin D (pCD) in HCC tissues. Knockdown of cathepsin D (CD) expression mediated by siRNA significantly inhibited the in vitro invasion of two HCC cell lines, SNU449 and SNU473, which normally secrete high‐levels of CD. Prefractionation using individual lectins demonstrated an elevation in ConA‐binding glycoforms of proCD and CD in HCC tissues. In the serum of HCC patients, “ConA‐binding proCD” (ConA‐pCD) is significantly increased in concentration and this increase is comprised of several distinct upregulated acidic isoforms (pI 4.5–5.5). Receiver operating characteristic analysis showed that the sensitivity and specificity of serum ConA‐pCD for HCC diagnosis were 85% and 80%, respectively. This is the first report that serum ConA‐pCD is increased significantly in HCC and is potentially useful as a serological biomarker for diagnosis of HCC.

Transcriptional intermediary factor 1γ binds to the anaphase-promoting complex/cyclosome and promotes mitosis.

The anaphase-promoting complex/cyclosome (APC/C) is an ubiquitin ligase that functions during mitosis. Here we identify the transcriptional regulator, transcriptional intermediary factor 1γ, TIF1γ, as an APC/C-interacting protein that regulates APC/C function. TIF1γ is not a substrate for APC/C-dependent ubiquitylation but instead, associates specifically with the APC/C holoenzyme and Cdc20 to affect APC/C activity and progression through mitosis. RNA interference studies indicate that TIF1γ knockdown results in a specific reduction in APC/C ubiquitin ligase activity, the stabilization of APC/C substrates, and an increase in the time taken for cells to progress through mitosis from nuclear envelope breakdown to anaphase. TIF1γ knockdown cells are also characterized by the inappropriate presence of cyclin A at metaphase, and an increase in the number of cells that fail to undergo metaphase-to-anaphase transition. Expression of a small interfering RNA-resistant TIF1γ species relieves the mitotic phenotype imposed by TIF1γ knockdown and allows for mitotic progression. Binding studies indicate that TIF1γ is also a component of the APC/C-mitotic checkpoint complex (MCC), but is not required for MCC dissociation from the APC/C once the spindle assembly checkpoint (SAC) is satisfied. TIF1γ inactivation also results in chromosome misalignment at metaphase and SAC activation; inactivation of the SAC relieves the mitotic block imposed by TIF1γ knockdown. Together these data define novel functions for TIF1γ during mitosis and suggest that a reduction in APC/C ubiquitin ligase activity promotes SAC activation.