143 research outputs found
The effect of timing and composition of gestational weight gain in obese pregnant women on infant birth weight: A prospective cohort study.
Introduction: CK2 is a protein kinase implicated in several essential cellular
processes, over-expressed in cancer and described to regulate insulin
signaling cascade. Recently CK2 has been described to negatively regulate
thermogenesis (Shinoda K et al, 2015, Cell Metabolism) and to inhibit
insulin release (Rossi M et al, 2015, PNAS). Nevertheless, the role of CK2
in adipose tissue (AT) and its involvement in human obesity development
and therapy has been poorly investigated.
Methods: Our multi-disciplinary team performed biochemical analysis of
signaling pathways by WB and in vitro kinase activity assays, and glucose
handling studies using glucose uptake assay and IF in adipocyte cultures
and glucose and insulin tolerance test in mice. Moreover we quantify CK2
expression/activity in human AT specimens of 27 obese patients, clinically
characterized, in 12 obese patients underwent relevant weight loss and 11
normal-weight controls.
Results: We proved that CK2 amount and activity were not influenced
by insulin stimulation and that CK2 activity was efficiently inhibited by
specific inhibitors, structurally unrelated. We worked with CX-4945, a
CK2 inhibitor currently used in cancer clinical trials, using the minimal
concentration (2.5 \u192
dM) and pre-treatment time (1hr) able to efficiently
inhibit CK2 activity, avoiding any cytotoxic effect. Pharmacological
inhibition of CK2 did not significantly affect in vitro adipogenic differentiation
or expression profiling of mature adipocytes. Conversely, we
showed that in human and murine adipocytes CK2-inhibition decreases
the insulin-induced glucose uptake by counteracting Akt-signaling and
GLUT4-translocation to the plasma membrane. We compared CK2 expression
and activity in different mouse tissues highlighted that white
skeletal muscle fibres and liver contained the highest quantity of this kinase.
CK2 was expressed more in brown AT than in white AT depots. We
show that CK2 promotes insulin-signaling in mouse AT, liver and skeletal
muscle and that in vivo acute treatment with CX-4945 impairs glucose-
tolerance in mice. Studies in tissues of ob/ob and db/db mice highlights
an up-regulation of CK2 expression and activity only in WAT. CK2
hyper-activation is strongly evident also in SAT and VAT of obese patients
and weight loss obtained by bariatric surgery or hypocaloric diet reverts
CK2 up-regulation to normal level.
Conclusion: We show that CK2 is involved in insulin sensitivity, glucose
handling and remodeling of WAT. Moreover we identify CK2 hyper-activation
as a hallmark of human obesity, suggesting a new potential therapeutic
target for metabolic diseases
Molecular targets for the protodynamic action of cis-urocanic acid in human bladder carcinoma cells
<p>Abstract</p> <p>Background</p> <p>cis-urocanic acid (cis-UCA) is an endogenous amino acid metabolite capable of transporting protons from the mildly acidic extracellular medium into the cell cytosol. The resulting intracellular acidification suppresses many cellular activities. The current study was aimed at characterizing the molecular mechanisms underlying cis-UCA-mediated cytotoxicity in cultured cancer cells.</p> <p>Methods</p> <p>5367 bladder carcinoma cells were left untreated or treated with cis-UCA. Cell death was assessed by measuring caspase-3 activity, mitochondrial membrane polarization, formation and release of cytoplasmic histone-associated DNA fragments, and cellular permeabilization. Cell viability and metabolic activity were monitored by colorimetric assays. Nuclear labelling was used to quantify the effects of cis-UCA on cell cycle. The activity of the ERK and JNK signalling pathways was studied by immunoblotting with specific antibodies. Phosphatase activity in cis-UCA-treated cells was determined by assay kits measuring absorbance resulting from the dephosphorylation of an artificial substrate. All statistical analyses were performed using the two-way Student's t-test (p < 0.05).</p> <p>Results</p> <p>Here we report that treatment of the 5637 human bladder carcinoma cells with 2% cis-UCA induces both apoptotic and necrotic cell death. In addition, metabolic activity of the 5637 cells is rapidly impaired, and the cells arrest in cell cycle in response to cis-UCA. Importantly, we show that cis-UCA promotes the ERK and JNK signalling pathways by efficiently inhibiting the activity of serine/threonine and tyrosine phosphatases.</p> <p>Conclusions</p> <p>Our studies elucidate how cis-UCA modulates several cellular processes, thereby inhibiting the proliferation and survival of bladder carcinoma cells. These anti-cancer effects make cis-UCA a potential candidate for the treatment of non-muscle invasive bladder carcinoma.</p
The Legionella effector WipB is a translocated Ser/Thr phosphatase that targets the host lysosomal nutrient sensing machinery
Legionella pneumophila infects human alveolar macrophages and is responsible for Legionnaire’s disease, a severe form of pneumonia. L. pneumophila encodes more than 300 putative effectors, which are translocated into the host cell via the Dot/Icm type IV secretion system. These effectors highjack the host’s cellular processes to allow bacterial intracellular growth and replication. Here we adopted a multidisciplinary approach to investigate WipB, a Dot/Icm effector of unknown function. The crystal structure of the N-terminal domain at 1.7 Å resolution comprising residues 25 to 344 revealed that WipB harbours a Ser/Thr phosphatase domain related to the eukaryotic phospho-protein phosphatase (PPP) family. The C-terminal domain (residues 365–524) is sufficient to pilot the effector to acidified LAMP1-positive lysosomal compartments, where WipB interacts with the v-ATPase and the associated LAMTOR1 phosphoprotein, key components of the lysosomal nutrient sensing (LYNUS) apparatus that controls the mammalian target of rapamycin (mTORC1) kinase complex at the lysosomal surface. We propose that WipB is a lysosome-targeted phosphatase that modulates cellular nutrient sensing and the control of energy metabolism during Legionella infection
Switches, Excitable Responses and Oscillations in the Ring1B/Bmi1 Ubiquitination System
In an active, self-ubiquitinated state, the Ring1B ligase monoubiquitinates histone H2A playing a critical role in Polycomb-mediated gene silencing. Following ubiquitination by external ligases, Ring1B is targeted for proteosomal degradation. Using biochemical data and computational modeling, we show that the Ring1B ligase can exhibit abrupt switches, overshoot transitions and self-perpetuating oscillations between its distinct ubiquitination and activity states. These different Ring1B states display canonical or multiply branched, atypical polyubiquitin chains and involve association with the Polycomb-group protein Bmi1. Bistable switches and oscillations may lead to all-or-none histone H2A monoubiquitination rates and result in discrete periods of gene (in)activity. Switches, overshoots and oscillations in Ring1B catalytic activity and proteosomal degradation are controlled by the abundances of Bmi1 and Ring1B, and the activities and abundances of external ligases and deubiquitinases, such as E6-AP and USP7
Inhibition of tyrosine protein kinases by the antineoplastic agent adriamycin.
Adriamycin, a lipid-interacting anti-cancer agent, was found to inhibit the phosphorylation of polyGlu/Tyr (4:1) by tyrosine protein kinases either from spleen or expressed by the oncogene of Abelson murine leukemia virus. The dose dependent inhibition by adriamycin is accounted for by competition for the ATP binding site, but it is also deeply influenced by the nature and concentration of the phosphorylatable substrate, suggesting multiple interactions with the enzyme. The phosphorylation at tyrosine residues of cytosolic proteins from cells transformed by Abelson leukemia virus and the autophosphorylation of tyrosine protein kinases are also inhibited by adriamycin. Unlike tyrosine protein kinases most serine/threonine specific protein kinases, with the notable exception of protein kinase-C, appear to be relatively insensitive to adriamycin
Distribution of protein disulphide isomerase in rat liver mitochondria.
Here we report the localization of protein disulphide isomerase (PDI) in the mitochondrial compartments, comparing it with that of thioredoxin reductase. The latter enzyme is present mostly in the matrix, whereas PDI is located at the level of the outer membrane. We characterize the different submitochondrial fractions with specific marker enzymes. PDI, whether isolated from whole mitochondria or from purified outer membranes, exhibits the same electrophoretic mobility, indicating identical molecular masses. Moreover, immunoblot analysis with monoclonal anti-PDI antibody shows immunoreactivity only with the microsomal PDI, indicating the specificity of the mitochondrial isoform. The significance of these findings is discussed with reference to the potential role of PDI and thioredoxin reductase in regulating the mitochondrial functions dependent on the thiol-disulphide transition
Inhibitory effect of prostacyclin and nitroprusside on type IIB von Willebrand factor-promoted platelet activation.
Abstract
Von Willebrand disease (vWD) of type IIB is a hereditary haemorrhagic disorder characterised by an excessive interaction of von Willebrand factor (vWF) with the platelet receptor GPIb which promotes platelet activation and aggregation through a phospholipase A2-mediated release of arachidonic acid. The present report shows that prostacyclin and nitroprusside, vasodilator-compounds that enhance the cAMP and cGMP concentration respectively, cause a drastic inhibition of the type IIB vWF-induced platelet responses including increase of cytosolic Ca2+ concentration, phosphorylation of pleckstrin (47 kDa) and myosin light chain (20 kDa), secretion of ATP and serotonin, and aggregation parallel to a decrease of arachidonic acid release. Type IIB vWF also elicits tyrosine phosphorylation of proteins with apparent molecular mass of 60, 74, 82 and 130 kDa. Prostacyclin, which induces per se tyrosine-phosphorylation of proteins of about 38 and 45 kDa, inhibits drastically the type IIB vWF-promoted tyrosine-phosphorylation of the 74 kDa protein while inhibits slightly that of 60 kDa band. The protein tyrosine-kinase inhibitor genistein causes a little decrease in the type IIB vWF-induced release of arachidonic acid. It is concluded that the inhibition exerted by prostacyclin and nitroprusside on type IIB vWF-elicited platelet activation seems to be largely ascribable to prevention of the phospholipase A2 activation with the ensuing decrease of the subsequent protein tyrosine phosphorylation
Phosphorylated residues as specificity determinants for an acidophilic protein tyrosine kinase. A study with src and cdc2 derived phosphopeptides.
none6noneDonella-Deana A;Marin O;Brunati AM;Cesaro L;Piutti C;Pinna LADonella, Arianna; Marin, Oriano; Brunati, ANNA MARIA; Cesaro, Luca; Piutti, C; Pinna, Lorenz
- …