Glycodelin-A interferes with IL-2/IL-2R signalling to induce cell growth arrest, loss of effector functions and apoptosis in T-lymphocytes

The progesterone-regulated glycoprotein glycodelin-A (GdA), secreted by the decidualized endometrium at high concentrations in primates, inhibits the maternal immune response against fetal antigens and thereby contributes to the tolerance of the semi-allogenic fetus during a normal pregnancy. Our earlier studies demonstrated the ability of GdA to induce an intrinsic apoptotic cascade in CD4 T-lymphocytes and suppress the cytolytic effector function of CD8 T-lymphocytes. In this report, we investigated further into the mechanism of action of GdA controlling perforin and granzyme B expression in CD8 T-lymphocytes and the mechanism of action of GdA leading to lymphocyte death. Flow cytometry analysis was performed to check for the surface expression of interleukin-2 receptor (IL-2R) and intracellular eomesodermin (Eomes) in activated T-lymphocytes, whereas quantitative RTPCR analysis was used to find out their mRNA profile upon GdA treatment. Western analysis was carried out to confirm the protein level of Bax and Bcl-2. GdA reduces the surface expression of the high-affinity IL-2R complex by down-regulating the synthesis of IL-2R (CD25). This disturbs the optimal IL-2 signalling and decreases the Eomes expression, which along with IL-2 directly regulates perforin and granzymes expression. Consequently, the CD8 T-lymphocytes undergo growth arrest and are unable to mature into competent cytotoxic T-lymphocytes. In the CD4 T-lymphocytes, growth factor IL-2 deprivation leads to proliferation inhibition, decreased Bcl-2/enhanced Bax expression, culminating in mitochondrial stress and cell death. GdA spurs cell cycle arrest, loss of effector functions and apoptosis in different T-cell subsets by making T-lymphocytes unable to respond to IL-2.

Serine/Threonine/Tyrosine Protein Kinase Phosphorylates Oleosin, a Regulator of Lipid Metabolic Functions

Plant oils are stored in oleosomes or oil bodies, which are surrounded by a monolayer of phospholipids embedded with oleosin proteins that stabilize the structure. Recently, a structural protein, Oleosin3 (OLE3), was shown to exhibit both monoacylglycerol acyltransferase and phospholipase A(2) activities. The regulation of these distinct dual activities in a single protein is unclear. Here, we report that a serine/threonine/tyrosine protein kinase phosphorylates oleosin. Using bimolecular fluorescence complementation analysis, we demonstrate that this kinase interacts with OLE3 and that the fluorescence was associated with chloroplasts. Oleosin-green fluorescent protein fusion protein was exclusively associated with the chloroplasts. Phosphorylated OLE3 exhibited reduced monoacylglycerol acyltransferase and increased phospholipase A(2) activities. Moreover, phosphatidylcholine and diacylglycerol activated oleosin phosphorylation, whereas lysophosphatidylcholine, oleic acid, and Ca2+ inhibited phosphorylation. In addition, recombinant peanut (Arachis hypogaea) kinase was determined to predominantly phosphorylate serine residues, specifically serine-18 in OLE3. Phosphorylation levels of OLE3 during seed germination were determined to be higher than in developing peanut seeds. These findings provide direct evidence for the in vivo substrate selectivity of the dual-specificity kinase and demonstrate that the bifunctional activities of oleosin are regulated by phosphorylation.

Primary Microcephaly Gene MCPH1 Shows Signatures of Tumor Suppressors and Is Regulated by miR-27a in Oral Squamous Cell Carcinoma

Mutations in the MCPH1 (microcephalin 1) gene, located at chromosome 8p23.1, result in two autosomal recessive disorders: primary microcephaly and premature chromosome condensation syndrome. MCPH1 has also been shown to be downregulated in breast, prostate and ovarian cancers, and mutated in 1/10 breast and 5/41 endometrial tumors, suggesting that it could also function as a tumor suppressor (TS) gene. To test the possibility of MCPH1 as a TS gene, we first performed LOH study in a panel of 81 matched normal oral tissues and oral squamous cell carcinoma (OSCC) samples, and observed that 14/71 (19.72%) informative samples showed LOH, a hallmark of TS genes. Three protein truncating mutations were identified in 1/15 OSCC samples and 2/5 cancer cell lines. MCPH1 was downregulated at both the transcript and protein levels in 21/41 (51.22%) and 19/25 (76%) OSCC samples respectively. A low level of MCPH1 promoter methylation was also observed in 4/40 (10%) tumor samples. We further observed that overexpression of MCPH1 decreased cellular proliferation, anchorage-independent growth in soft agar, cell invasion and tumor size in nude mice, indicating its tumor suppressive function. Using bioinformatic approaches and luciferase assay, we showed that the 3'-UTR of MCPH1 harbors two non-overlapping functional seed regions for miR-27a which negatively regulated its level. The expression level of miR-27a negatively correlated with the MCPH1 protein level in OSCC. Our study indicates for the first time that, in addition to its role in brain development, MCPH1 also functions as a tumor suppressor gene and is regulated by miR-27a.

USP1 deubiquitinase: cellular functions, regulatory mechanisms and emerging potential as target in cancer therapy

12 p.

Developmentally regulated transcription factors in Drosophila melanogaster

During early stages of Drosophila development the heat shock response cannot be induced. It is reasoned that the adverse effects on cell cycle and cell growth brought about by Hsp70 induction must outweigh the beneficial aspects of Hsp70 induction in the early embryo. Although the Drosophila heat shock transcription factor (dHSF) is abundant in the early embryo, it does not enter the nucleus in response to heat shock. In older embryos and in cultured cells the factor is localized within the nucleus in an apparent trimeric structure that binds DNA with high affinity. The domain responsible for nuclear localization upon stress resides between residues 390 and 420 of the dHSF. Using that domain as bait in a yeast two-hybrid system we now report the identification and cloning of a nuclear transport protein Drosophila karyopherin-α3(dKap- α3). Biochemical methods demonstrate that the dKap-α3 protein binds specifically to the dHSF's nuclear localization sequence (NLS). Furthermore, the dKap-α3 protein does not associate with NLSs that contain point mutations which are not transported in vivo. Nuclear docking studies also demonstrate specific nuclear targeting of the NLS substrate by dKap-α3.Consistant with previous studies demonstrating that early Drosophila embryos are refractory to heat shock as a result of dHSF nuclear exclusion, we demonstrate that the early embryo is deficient in dKap-α3 protein through cycle 12. From cycle 13 onward the transport factor is present and the dHSF is localized within the nucleus thus allowing the embryo to respond to heat shock.

The pair-rule gene fushi tarazu (ftz) is a well-studied zygotic segmentation gene that is necessary for the development of the even-numbered parasegments in Drosophila melanogastor. During early embryogenesis, ftz is expressed in a characteristic pattern of seven stripes, one in each of the even-numbered parasegments. With a view to understand how ftz is transcriptionally regulated, cDNAs that encode transcription factors that bind to the zebra element of the ftz promoter have been cloned. Chapter Ill reports the cloning and characterization of the eDNA encoding zeb-1 (zebra element binding protein), a novel steroid receptor-like molecule that specifically binds to a key regulatory element of the ftz promoter. In transient transfection assays employing Drosophila tissue culture cells, it has been shown that zeb-1 as well as a truncated zeb-1 polypeptide (zeb480) that lacks the putative ligand binding domain function as sequencespecific trans-activators of the ftz gene.

The Oct factors are members of the POU family of transcription factors that are shown to play important roles during development in mammals. Chapter IV reports the eDNA cloning and expression of a Drosophila Oct transcription factor. Whole mount in-situ hybridization experiments revealed that the spatial expression patterns of this gene during embryonic development have not yet been observed for any other gene. In early embryogenesis, its transcripts are transiently expressed as a wide uniform band from 20-40% of the egg length, very similar to that of gap genes. This pattern progressively resolves into a series of narrower stripes followed by expression in fourteen stripes. Subsequently, transcripts from this gene are expressed in the central nervous system and the brain. When expressed in the yeast Saccharomyces cerevisiae, this Drosophila factor functions as a strong, octamer-dependent activator of transcription. The data strongly suggest possible functions for the Oct factor in pattern formation in Drosophila that might transcend the boundaries of genetically defined segmentation genes.

Cloning and expression of glucose regulated protein 78 (GRP78) in Fenneropenaeus chinensis

GRP78 (78 kDa glucose-regulated protein), also known as BiP (immunoglobulin heavy-chain-binding protein), is an essential regulator of endoplasmic reticulum (ER) homeostasis because of its multiple functions in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. In this report, we cloned the full length cDNA of GRP78 (FcGRP78) from Chinese shrimp Fenneropenaeus chinensis. This cDNA revealed a 2,325 bp with 1,968 bp open reading frame encoding 655 amino acids. This is the first reported GRP78 gene in Crustacea. The deduced amino acid sequence of FcGRP78 shared high identity with previously reported insect GRP78s: 86, 87 and 85% identity with GRP78s of Drosophila melanogaster, Aedes aegypti and Bombyx mori, respectively. Northern blot analysis shows that FcGRP78 is ubiquitously expressed in tissues of shrimp. Heat shock at 35A degrees C significantly enhanced the expression of FcGRP78 at the first hour, reached the maximum at 4 h post heat shock, dropped after that and resumed to the normal level until 48 h of post recovery at 25A degrees C. Additionally, differential expression of FcGRP78 was detected in haemocytes, hepatopancreas and lymphoid organ when shrimp were challenged by white spot syndrome virus (WSSV). We inferred that FcGRP78 may play important roles in chaperoning, protein folding and immune function of shrimp.

A novel syndrome of severe neutrophil dysfunction: unresponsiveness confined to chemotaxin-induced functions.

We have identified a patient with a number of neutrophil dysfunctions. The patient was a female baby who lived for 8 months. During her life, she developed severe bacterial infections and showed omphalitis, impaired wound healing, and a pronounced leukocytosis. She was not a patient with leukocyte adhesion deficiency, because all leukocyte CD18 complex proteins were expressed at normal levels. Yet, neutrophil polarization and chemotaxis to platelet-activating factor, leukotriene B4, or formyl-methionyl-leucyl-phenylalanine (FMLP) were completely absent. We found a strong defect in actin polymerization in response to chemotactic stimuli, but only a retarded or even normal reaction with other stimuli. This indicates that the cellular dysfunctions were not due to an intrinsic defect in actin metabolism. Instead, the regulation of actin polymerization with chemotactic stimuli seemed to be defective. We concentrated on FMLP-induced responses in the patient's neutrophils. Functions dependent on activation of complement receptor type 3, such as aggregation or adherence to endothelial cells, were normally induced. Binding to serum-coated coverslips was normal in cell number; however, spreading was not observed. Exocytosis from the specific granules was readily induced. In contrast, FMLP failed to induce a respiratory burst activity or degranulation of the azurophil granules. FMLP induced a normal increase in free intracellular Ca2+, but a decreased formation of diglycerides (especially the 1-O-alkyl,2-acyl compounds). Thus, we have described a patient whose neutrophils show a severe defect in functional activation via chemotaxin receptors, resulting in a selective absence of NADPH oxidase activity, exocytosis from the azurophil granules, and actin polymerization. Our findings show that actin polymerization for neutrophil spreading and locomotion is regulated differently from that for phagocytosis. Also, the release of azurophil and specific granule contents is clearly shown to be regulated in a different way.

Unfolded protein response genes regulated by CED-1 are required for Caenorhabditis elegans innate immunity.

The endoplasmic reticulum stress response, also known as the unfolded protein response (UPR), has been implicated in the normal physiology of immune defense and in several disorders, including diabetes, cancer, and neurodegenerative disease. Here, we show that the apoptotic receptor CED-1 and a network of PQN/ABU proteins involved in a noncanonical UPR response are required for proper defense to pathogen infection in Caenorhabditis elegans. A full-genome microarray analysis indicates that CED-1 functions to activate the expression of pqn/abu genes. We also show that ced-1 and pqn/abu genes are required for the survival of C. elegans exposed to live Salmonella enterica, and that overexpression of pqn/abu genes confers protection against pathogen-mediated killing. The results indicate that unfolded protein response genes, regulated in a CED-1-dependent manner, are involved in the C. elegans immune response to live bacteria.

Identification of late larval stage developmental checkpoints in Caenorhabditis elegans regulated by insulin/IGF and steroid hormone signaling pathways.

Organisms in the wild develop with varying food availability. During periods of nutritional scarcity, development may slow or arrest until conditions improve. The ability to modulate developmental programs in response to poor nutritional conditions requires a means of sensing the changing nutritional environment and limiting tissue growth. The mechanisms by which organisms accomplish this adaptation are not well understood. We sought to study this question by examining the effects of nutrient deprivation on Caenorhabditis elegans development during the late larval stages, L3 and L4, a period of extensive tissue growth and morphogenesis. By removing animals from food at different times, we show here that specific checkpoints exist in the early L3 and early L4 stages that systemically arrest the development of diverse tissues and cellular processes. These checkpoints occur once in each larval stage after molting and prior to initiation of the subsequent molting cycle. DAF-2, the insulin/insulin-like growth factor receptor, regulates passage through the L3 and L4 checkpoints in response to nutrition. The FOXO transcription factor DAF-16, a major target of insulin-like signaling, functions cell-nonautonomously in the hypodermis (skin) to arrest developmental upon nutrient removal. The effects of DAF-16 on progression through the L3 and L4 stages are mediated by DAF-9, a cytochrome P450 ortholog involved in the production of C. elegans steroid hormones. Our results identify a novel mode of C. elegans growth in which development progresses from one checkpoint to the next. At each checkpoint, nutritional conditions determine whether animals remain arrested or continue development to the next checkpoint.

MicroRNA-34c inversely couples the biological functions of the runt-related transcription factor RUNX2 and the tumor suppressor p53 in osteosarcoma

Osteosarcoma (OS) is a primary bone tumor that is most prevalent during adolescence. RUNX2, which stimulates differentiation and suppresses proliferation of osteoblasts, is deregulated in OS. Here, we define pathological roles of RUNX2 in the etiology of OS and mechanisms by which RUNX2 expression is stimulated. RUNX2 is often highly expressed in human OS biopsies and cell lines. Small interference RNA (siRNA)-mediated depletion of RUNX2 inhibits growth of U2OS OS cells. RUNX2 levels are inversely linked to loss of p53 (which predisposes to OS) in distinct OS cell lines and osteoblasts. RUNX2 protein levels decrease upon stabilization of p53 with the MDM2 inhibitor Nutlin-3. Elevated RUNX2 protein expression is post-transcriptionally regulated and directly linked to diminished expression of several validated RUNX2 targeting microRNAs (miRNAs) in human OS cells compared to mesenchymal progenitor cells. The p53-dependent miR-34c is the most significantly down-regulated RUNX2 targeting miRNA in OS. Exogenous supplementation of miR-34c markedly decreases RUNX2 protein levels, while 3UTR reporter assays establish RUNX2 as a direct target of miR-34c in OS cells. Importantly, Nutlin-3 mediated stabilization of p53 increases expression of miR-34c and decreases RUNX2. Thus, a novel RUNX2-p53-miR34 network controls cell growth of osseous cells and is compromised in OS.

Functional and pharmacological characterization of volume-regulated anion channels in human normal and cystic fibrosis bronchial and nasal epithelial cells

Volume-regulated anion channels (VRACs) are widely present in various cell types and have important functions ranging from regulatory volume decrease to control of cell proliferation and apoptosis. Here we aimed to compare the biophysical features and pharmacological profiles of VRAC currents in healthy and cystic fibrosis (CF) respiratory epithelial cells in order to characterize these currents both functionally and pharmacologically. Whole-cell electrophysiology was used to characterize the VRAC current in normal (16HBE14o-; HBE) and CF cell lines (CFBE14o-; CFBE), as well as in native human nasal epithelial cells. Application of hypotonic solution produced current responses of similar sizes in both HBE and CFBE cells. Biophysical properties of VRACs, such as instantaneous activation and deactivation upon voltage step, some inactivation at potentials positive to 40 mV and outwardly-rectifying I-V curves, were indistinguishable in both cell types. Extensive pharmacological analysis of the currents revealed a similar pharmacological profile in response to three blockers--NPPB, DCPIB and DIDS. Native primary human nasal epithelial cells from both healthy and CF volunteers also showed typical VRAC responses of comparable sizes. VRACs in these cells were more sensitive to external solution hypotonicity compared to HBE and CFBE cells. In all cell types studied robust VRAC currents could be induced at constant cell volume by G-protein activation with GTPγS infusion. This study provides the first extensive comparative functional and pharmacological analysis of VRAC currents in normal and CF airway epithelial cells and shows that VRACs are unimpaired molecularly or functionally in CF.

Nuclear translocation and functions of growth factor receptors

Cellular signal transduction in response to environmental signals involves a relay of precisely regulated signal amplifying and damping events. A prototypical signaling relay involves ligands binding to cell surface receptors and triggering the activation of downstream enzymes to ultimately affect the subcellular distribution and activity of DNA-binding proteins that regulate gene expression. These so-called signal transduction cascades have dominated our view of signaling for decades. More recently evidence has accumulated that components of these cascades can be multifunctional, in effect playing a conventional role for example as a cell surface receptor for a ligand whilst also having alternative functions for example as transcriptional regulators in the nucleus. This raises new challenges for researchers. What are the cues/triggers that determine which role such proteins play? What are the trafficking pathways which regulate the spatial distribution of such proteins so that they can perform nuclear functions and under what circumstances are these alternative functions most relevant?

PP1 interactomes as a means of characterizing protein functions

A maioria das funções celulares, incluindo expressão de genes, crescimento e proliferação celulares, metabolismo, morfologia, motilidade, comunicação intercelular e apoptose, é regulada por interações proteína-proteína (IPP). A célula responde a uma variedade de estímulos, como tal a expressão de proteínas é um processo dinâmico e os complexos formados são constituídos transitoriamente mudando de acordo com o seu ciclo funcional, adicionalmente, muitas proteínas são expressas de uma forma dependente do tipo de célula. Em qualquer instante a célula pode conter cerca de centenas de milhares de IPPs binárias, e encontrar os companheiros de interação de uma proteína é um meio de inferir a sua função. Alterações em redes de IPP podem também fornecer informações acerca de mecanismos de doença. O método de identificação binário mais frequentemente usado é o sistema Dois Hibrido de Levedura, adaptado para rastreio em larga escala. Esta metodologia foi aqui usada para identificar os interactomas específicos de isoforma da Proteína Fosfatase 1 (PP1), em cérebro humano. A PP1 é uma proteína fosfatase de Ser/Thr envolvida numa grande variedade de vias e eventos celulares. É uma proteína conservada codificada por três genes, que originam as isoformas α, β, e γ, com a última a originar γ1 e γ2 por splicing alternativo. As diferentes isoformas da PP1 são reguladas pelos companheiros de interação – proteínas que interagem com a PP1 (PIPs). A natureza modular dos complexos da PP1, bem como a sua associação combinacional, gera um largo reportório de complexos reguladores e papéis em circuitos de sinalização celular. Os interactomas da PP1 específicos de isofoma, em cérebro, foram aqui descritos, com um total de 263 interações identificadas e integradas com os dados recolhidos de várias bases de dados de IPPs. Adicionalmente, duas PIPs foram selecionadas para uma caracterização mais aprofundada da interação: Taperina e Sinfilina-1A. A Taperina é uma proteína ainda pouco descrita, descoberta recentemente como sendo uma PIP. A sua interação com as diferentes isoformas da PP1 e localização celulares foram analisadas. Foi descoberto que a Taperina é clivada e que está presente no citoplasma, membrana e núcleo e que aumenta os níveis de PP1, em células HeLa. Na membrana ela co-localiza com a PP1 e a actina e uma forma mutada da Taperina, no motivo de ligação à PP1, está enriquecida no núcleo, juntamente com a actina. Mais, foi descoberto que a Taperina é expressa em testículo e localiza-se na região acrossómica da cabeça do espermatozoide, uma estrutura onde a PP1 e a actina estão também presentes. A Sinfilina-1A, uma isoforma da Sinfilina-1, é uma proteína com tendência para agregar e tóxica, envolvida na doença de Parkinson. Foi mostrado que a Sinfilina-1A liga às isoformas da PP1, por co-transformação em levedura, e que mutação do seu motivo de ligação à PP1 diminuiu significativamente a interação, num ensaio de overlay. Quando sobre-expressa em células Cos-7, a Sinfilina-1A formou corpos de inclusão onde a PP1 estava presente, no entanto a forma mutada da Sinfilina-1A também foi capaz de agregar, indicando que a formação de inclusões não foi dependente de ligação à PP1. Este trabalho dá uma nova perspetiva dos interactomas da PP1, incluindo a identificação de dezenas de companheiros de ligação específicos de isoforma, e enfatiza a importância das PIPs, não apenas na compreensão das funções celulares da PP1 mas também, como alvos de intervenção terapêutica.

Nuclear receptor Rev-erb alpha (Nr1d1) functions in concert with Nr2e3 to regulate transcriptional networks in the retina.

The majority of diseases in the retina are caused by genetic mutations affecting the development and function of photoreceptor cells. The transcriptional networks directing these processes are regulated by genes such as nuclear hormone receptors. The nuclear hormone receptor gene Rev-erb alpha/Nr1d1 has been widely studied for its role in the circadian cycle and cell metabolism, however its role in the retina is unknown. In order to understand the role of Rev-erb alpha/Nr1d1 in the retina, we evaluated the effects of loss of Nr1d1 to the developing retina and its co-regulation with the photoreceptor-specific nuclear receptor gene Nr2e3 in the developing and mature retina. Knock-down of Nr1d1 expression in the developing retina results in pan-retinal spotting and reduced retinal function by electroretinogram. Our studies show that NR1D1 protein is co-expressed with NR2E3 in the outer neuroblastic layer of the developing mouse retina. In the adult retina, NR1D1 is expressed in the ganglion cell layer and is co-expressed with NR2E3 in the outer nuclear layer, within rods and cones. Several genes co-targeted by NR2E3 and NR1D1 were identified that include: Nr2c1, Recoverin, Rgr, Rarres2, Pde8a, and Nupr1. We examined the cyclic expression of Nr1d1 and Nr2e3 over a twenty-four hour period and observed that both nuclear receptors cycle in a similar manner. Taken together, these studies reveal a novel role for Nr1d1, in conjunction with its cofactor Nr2e3, in regulating transcriptional networks critical for photoreceptor development and function.

MARCKS functions as a novel growth/tumour suppressor in cells of melanocyte origin

Protein kinase C (PKC) plays a pivotal role in modulating the growth of melanocytic cells in culture. We have shown previously that a major physiological substrate of PKC, the 80 kDa myristoylated alanine-rich C-kinase substrate (MARCKS), can be phosphorylated in quiescent, non-tumorigenic melanocytes exposed transiently to a biologically active phorbol ester, but cannot be phosphorylated in phorbol ester-treated, syngeneic malignant melanoma cells. Despite its ubiquitous distribution, the function of MARCKS in cell growth and transformation remains to be demonstrated clearly. We report here that MARCKS mRNA and protein levels are down-regulated significantly in the spontaneously derived murine B16 melanoma cell line compared with syngeneic normal Mel-ab melanocytes. In contrast, the tumourigenic v-Ha-ras-transfonned melan-ocytic line, LTR Ras 2, showed a high basal level of MARCKS phosphorylation which was not enhanced by treatment of cells with phorbol ester. Furthermore, protein levels of MARCKS in LTR Ras 2 cells were similar to those expressed in Mel-ab melanocytes. However, in four out of six murine tumour cell lines investigated, levels of MARCKS protein were barely detectable. Transfection of B16 cells with a plasmid containing the MARCKS cDNA in the sense orientation produced two neomycin-resistant clones displaying reduced proliferative capacity and decreased anchorage-independent growth compared with control cells. In contrast, transfection with the antisense MARCKS construct produced many colonies which displayed enhanced growth and transforming potential compared with control cells. Thus, MARCKS appears to act as a novel growth suppressor in the spontaneous transformation of cells of melanocyte origin and may play a more general role in the tumour progression of other carcinomas.