BBA - Molecular Cell Research (v.1783, #8)

The RCC1 superfamily: From genes, to function, to disease by Ouadah Hadjebi; Eduard Casas-Terradellas; Francesc R. Garcia-Gonzalo; Jose Luis Rosa (1467-1479).
The Regulator of Chromosome Condensation 1 (RCC1) was identified over 20  years ago as a critical cell cycle regulator. By analyzing its amino acid sequence, RCC1 was found to consist of seven homologous repeats of 51–68 amino acid residues, which were later shown to adopt a seven-bladed β-propeller fold. Since the initial identification of RCC1, a number of proteins have been discovered that contain one or more RCC1-like domains (RLDs). As we show here, these RCC1 superfamily proteins can be subdivided in five subgroups based on structural criteria. In recent years, a number of studies have been published regarding the functions of RCC1 superfamily proteins. From these studies, the emerging picture is that the RLD is a versatile domain which may perform many different functions, including guanine nucleotide exchange on small GTP-binding proteins, enzyme inhibition or interaction with proteins and lipids. Here, we review the available structural and functional data on RCC1 superfamily members, paying special attention to the human proteins and their involvement in disease.
Keywords: RCC1; RLD; GRF; GEF; HERC; RCBTB;

The secretion and maturation of prosaposin and procathepsin D are blocked in embryonic neural progenitor cells by Rosa Salvioli; Lucia Ricci-Vitiani; Massimo Tatti; Susanna Scarpa; Ruggero De Maria; Anna Maria Vaccaro (1480-1489).
The notion that prosaposin (Prosap) is likely involved in brain development and regeneration led us to explore its expression in stem/progenitor neural cells and its fate after cell differentiation. The expression of procathepsin–cathepsin D (proCath–Cath D), an endoprotease that plays an important role in the processing and sorting of Prosap, has been concomitantly examined. Our data evidenced that in embryonic human neural progenitor cells (eHNPCs) intact and high molecular weight intermediate forms of Prosap and intermediate forms of Cath D accumulated inside the cells, while the formation of saposins and mature Cath D was impaired. Furthermore, neither Prosap nor proCath D were secreted from eHNPCs. The block of the processing and secretion shared by Prosap and proCath D was overcome during the course of differentiation of eHNPCs into a mixed population of astrocytes and neuronal cells. Upon differentiation, large amounts of Prosap and proCath D were secreted from the cells, while saposins and mature Cath D were produced inside the cells. The dramatic accumulation of Prosap (an antiapoptotic factor) and reduction of mature Cath D (a proapoptotic factor) in the undifferentiated eHNPCs most likely play a role in the molecular mechanisms regulating the resistance to apoptotic signals of these cells and might represent a critically important issue in HNPCs biology.
Keywords: Prosaposin; Procathepsin D; Human neural stem cells; Post-translational modifications; Protein secretion; Alternative mRNA splicing;

Protein phosphatase 4 regulates apoptosis, proliferation and mutation rate of human cells by Mirna Mourtada-Maarabouni; Gwyn T. Williams (1490-1502).
The proteins which regulate apoptosis are of great importance both in normal cell biological processes and in the development of pathology in the diverse diseases which involve apoptosis dysfunction. The activity of many of these proteins is controlled by reversible phosphorylation, so that the relevant kinases and phosphatases play crucial roles in apoptosis control. Here we report the analysis of the role of the serine/threonine protein phosphatase, protein phosphatase 4, in controlling the apoptosis of HEK 293 T cells, using the complementary techniques of gene over-expression and down regulation through RNA interference. This analysis has demonstrated that PP4 regulates both apoptosis and proliferation in human cells and has also shown that the level of PP4 has a strong influence on gene mutation rate, which is crucial to oncogenesis. A parallel proteomic analysis has shown that the phosphorylation status of many relevant protein targets is affected by changes in PP4 and has focused attention particularly on the critical apoptosis regulators Bad and PEA-15. The phosphorylation of both of these proteins is increased when PP4 levels are suppressed, and is reduced when PP4 levels are increased, with striking consequences for the fate of the cell.
Keywords: Protein phosphatase 4; Apoptosis; Cell cycle; Bad; PEA-15; Tumour suppressor;

Stimulation- and palmitoylation-dependent changes in oligomeric conformation of serotonin 5-HT1A receptorsi by Fritz Kobe; Ute Renner; Andrew Woehler; Jakub Wlodarczyk; Ekaterina Papusheva; Guobin Bao; Andre Zeug; Diethelm W. Richter; Erwin Neher; Evgeni Ponimaskin (1503-1516).
In the present study we analyzed the oligomerization state of the serotonin 5-HT1A receptor and studied oligomerization dynamics in living cells. We also investigated the role of receptor palmitoylation in this process. Biochemical analysis performed in neuroblastoma N1E-115 cells demonstrated that both palmitoylated and non-palmitoylated 5-HT1A receptors form homo-oligomers and that the prevalent receptor species at the plasma membrane are dimers. A combination of an acceptor-photobleaching FRET approach with fluorescence lifetime measurements verified the interaction of CFP- and YFP-labeled wild-type as well as acylation-deficient 5-HT1A receptors at the plasma membrane of living cells. Using a novel FRET technique based on the spectral analysis we also confirmed the specific nature of receptor oligomerization. The analysis of oligomerization dynamics revealed that apparent FRET efficiency measured for wild-type oligomers significantly decreased in response to agonist stimulation, and our combined results suggest that this decrease was mediated by accumulation of FRET-negative complexes rather than by dissociation of oligomers to monomers. In contrast, the agonist-mediated decrease of FRET signal was completely abolished in oligomers composed by non-palmitoylated receptor mutants, demonstrating the importance of palmitoylation in modulation of the structure of oligomers.
Keywords: Oligomerization; Serotonin; G-protein coupled receptors; Palmitoylation; Foerster resonance energy transfer;

Role of NonO–histone interaction in TNFα-suppressed Prolyl-4-hydroxylase α1 by Cheng Zhang; Ming-Xiang Zhang; Ying H. Shen; Jared K. Burks; Xiao-Nan Li; Scott A. LeMaire; Koichi Yoshimura; Hiroki Aoki; Masunori Matsuzaki; Feng-Shuang An; David A. Engler; Risë K. Matsunami; Joseph S. Coselli; Yun Zhang; Xing Li Wang (1517-1528).
Inflammation is a key process in cardiovascular diseases. The extracellular matrix (ECM) of the vasculature is a major target of inflammatory cytokines, and TNFα regulates ECM metabolism by affecting collagen production. In this study, we have examined the pathways mediating TNFα-induced suppression of prolyl-4 hydroxylase alpha1 (P4Hα1), the rate-limiting isoform of P4H responsible for procollagen hydroxylation, maturation, and organization. Using human aortic smooth muscle cells, we found that TNFα activated the MKK4-JNK1 pathway, which induced histone (H) 4 lysine 12 acetylation within the TNFα response element in the P4Hα1 promoter. The acetylated-H4 then recruited a transcription factor, NonO, which, in turn, recruited HDACs and induced H3 lysine 9 deacetylation, thereby inhibiting transcription of the P4Hα1 promoter. Furthermore, we found that TNFα oxidized DJ-1, which may be essential for the NonO–P4Hα1 interaction because treatment with gene specific siRNA to knockout DJ-1 eliminated the TNFα-induced NonO–P4Hα1 interaction and its suppression. Our findings may be relevant to aortic aneurysm and dissection and the stability of the fibrous cap of atherosclerotic plaque in which collagen metabolism is important in arterial remodeling. Defining this cytokine-mediated regulatory pathway may provide novel molecular targets for therapeutic intervention in preventing plaque rupture and acute coronary occlusion.
Keywords: Inflammation; Histone; P4Hα1; TNFα; NonO; JNK1;

Knockdown of cytosolic 5′-nucleotidase II (cN-II) reveals that its activity is essential for survival in astrocytoma cells by Maria Giovanna Careddu; Simone Allegrini; Rossana Pesi; Marcella Camici; Mercedes Garcia-Gil; Maria Grazia Tozzi (1529-1535).
IMP preferring cytosolic 5′-nucleotidase (cN-II) is an ubiquitous nucleotide hydrolysing enzyme. The enzyme is widely distributed and its amino acid sequence is highly conserved among vertebrates. Fluctuations of cN-II activity have been associated with the pathogenesis of neurological disorders. The enzyme appears to be involved in the regulation of the intracellular availability of the purine precursor IMP and also of GMP and AMP, but the contribution of this activity and of its regulation to cell metabolism and to CNS cell functions remains uncertain. To address this issue, we used a vector based short hairpin RNA (shRNA) strategy to knockdown cN-II activity in human astrocytoma cells. Our results demonstrated that 53 h after transduction, cN-II mRNA was reduced to 17.9 ± 0.03% of control cells. 19 h later enzyme activity was decreased from 0.7 ± 0.026 mU/mg in control ADF cells to 0.45 ± 0.046 mU/mg, while cell viability (evaluated by the MTT reduction assay) decreased up to 0.59 ± 0.01 (fold vs control) and caspase 3 activity increased from 136 ± 5.8 pmol min− 1 mg− 1 in control cells to 639 ± 37.5 pmol min− 1 mg− 1 in silenced cells, thus demonstrating that cN-II is essential for cell survival. The decrease of enzyme activity causes apoptosis of the cultured cells without altering intracellular nucleotide and nucleoside concentration or energy charge. Since cN-II is highly expressed in tumour cells, our finding offers a new possible therapeutical approach especially against primary brain tumours such as glioblastoma, and to ameliorate chemotherapy against leukemia.
Keywords: cN-II; Cytosolic 5′-nucleotidase; Purine analogs; shRNA; RNAi; Apoptosis; ADF;

Direct protein–protein interaction of 11β-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the endoplasmic reticulum lumen by Atanas G. Atanasov; Lyubomir G. Nashev; Laurent Gelman; Balazs Legeza; Ragna Sack; Reto Portmann; Alex Odermatt (1536-1543).
Hexose-6-phosphate dehydrogenase (H6PDH) has been shown to stimulate 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1)-dependent local regeneration of active glucocorticoids. Here, we show that coexpression with H6PDH results in a dramatic shift from 11β-HSD1 oxidase to reductase activity without affecting the activity of the endoplasmic reticular enzyme 17β-HSD2. Immunoprecipitation experiments revealed coprecipitation of H6PDH with 11β-HSD1 but not with the related enzymes 11β-HSD2 and 17β-HSD2, suggesting a specific interaction between H6PDH and 11β-HSD1. The use of the 11β-HSD1/11β-HSD2 chimera indicates that the N-terminal 39 residues of 11β-HSD1 are sufficient for interaction with H6PDH. An important role of the N-terminus was indicated further by the significantly stronger interaction of 11β-HSD1 mutant Y18-21A with H6PDH compared to wild-type 11β-HSD1. The protein–protein interaction and the involvement of the N-terminus of 11β-HSD1 were confirmed by Far-Western blotting. Finally, fluorescence resonance energy transfer (FRET) measurements of HEK-293 cells expressing fluorescently labeled proteins provided evidence for an interaction between 11β-HSD1 and H6PDH in intact cells. Thus, using three different methods, we provide strong evidence that the functional coupling between 11β-HSD1 and H6PDH involves a direct physical interaction of the two proteins.
Keywords: 11beta-hydroxysteroid dehydrogenase; Hexose-6-phosphate dehydrogenase; Glucocorticoid; Endoplasmic reticulum; Protein interaction; Reductase;

Novel translocation responses of cytosolic phospholipase A2α fluorescent proteins by Rhonda E. Wooten; Mark C. Willingham; Larry W. Daniel; Christina C. Leslie; LeAnn C. Rogers; Susan Sergeant; Joseph T. O'Flaherty (1544-1550).
Cytosolic phospholipase A2 (cPLA2)α responds to the rise in cytosolic Ca2+ ([Ca2+]i) attending cell stimulation by moving to intracellular membranes, releasing arachidonic acid (AA) from these membranes, and thereby initiating the synthesis of various lipid mediators. Under some conditions, however, cPLA2α translocation occurs without any corresponding changes in [Ca2+]i. The signal for such responses has not been identified. Using confocal microscopy to track fluorescent proteins fused to cPLA2α or cPLA2α's C2 domain, we find that AA mimics Ca2+ ionophores in stimulating cPLA2α translocations to the perinuclear ER and to a novel site, the lipid body. Unlike the ionophores, AA acted independently of [Ca2+]i rises and did not translocate the proteins to the Golgi. AA's action did not involve its metabolism to eicosanoids or acylation into cellular lipids. Receptor agonists also stimulated translocations targeting lipid bodies. We propose that AA is a signal for Ca2+-independent cPLA2α translocation and that lipid bodies are common targets of cPLA2α and contributors to stimulus-induced lipid mediator synthesis.
Keywords: cPLA2α; C2 domain; Translocation; Arachidonic acid; Lipid body; Cellular calcium; Cancer cell;

High content analysis of γ-secretase activity reveals variable dominance of presenilin mutations linked to familial Alzheimer's disease by Cristina Florean; Enrico Zampese; Marion Zanese; Lucia Brunello; François Ichas; Francesca De Giorgi; Paola Pizzo (1551-1560).
γ-Secretase mediates the intramembranous proteolysis of amyloid precursor protein (APP), Notch and other cellular substrates and is considered a prime pharmacological target in the development of therapeutics for Alzheimer's disease (AD). We describe here an efficient, new, simple, sensitive and rapid assay to quantify γ-secretase activity in living cells by flow cytometry using two membrane-bound fluorescent probes, APP-GFP or C99-GFP, as substrates for γ-secretase. The principle of the assay is based on the fact that the soluble intracellular domain of GFP-tagged APP (AICD-GFP) is released from the membrane into the cytosol following γ-secretase cleavage. Using this feature, enzymatic activity of γ-secretase could be deduced from the extent of the membrane retention of the probe observed after plasma membrane permeabilization and washout of the cleaved fraction. By applying two well-known γ-secretase inhibitors (DAPT and L-685,458), we validated our assay showing that the positional GFP-based probes for γ-secretase activity behave properly when expressed in different cell lines, providing the basis for the further development of a high-throughput and high content screening for AD targeted drug discovery. Moreover, by co-expression of different familial AD-linked mutated forms of presenilin – the key component of the γ-secretase complex – in cells devoid of any endogenous γ-secretase, our method allowed us to evaluate in situ the contribution of different presenilin variants to the modulation of the enzyme.
Keywords: γ-Secretase assay; Alzheimer's disease; Presenilin; High content analysis; Recombinant probe; Automated flow cytometry;

Corrigendum to “Nuclear import of human sexual regulator DMRT1 is mediated by importin-β” [Biochim. Biophys. Acta 1773 (2007) 804–813] by Ming Ying; Bo Chen; Yihao Tian; Yu Hou; Qin Li; Xuan Shang; Jinhua Sun; Hanhua Cheng; Rongjia Zhou (1561).