Analysis of TAp73-dependent signaling via omics technologies.

Transactivation-proficient (TA) p73 is a transcription factor belonging to the p53 family, which regulates a variety of biological processes, including neurogenesis, differentiation, apoptosis, and DNA damage checkpoint response. In the present study, we adopted multiple Omics approaches, based upon the simultaneous application of metabolomics, lipidomics, and proteomics, in order to dissect the intracellular pathways activated by p73. As cellular model, we utilized a clone of the human osteosarcoma SAOS-2 cell line that allows the expression of TAp73α in an inducible manner. We found that TAp73α promoted mitochondrial activity (accumulation of metabolic intermediates and up-regulation of proteins related to the Krebs cycle), boosted glutathione homeostasis, increased arginine-citrulline-NO metabolism, altered purine synthesis, and promoted the pentose phosphate pathway toward NADPH accumulation for reducing and biosynthetic purposes. Indeed, lipid metabolism was driven toward the accumulation and oxidation of long-chain fatty acids with pro-apoptotic potential. In parallel, the expression of TAp73α was accompanied by the dephosphorylation of key proteins of the mitotic spindle assembly checkpoint. In conclusion, the obtained results confirm existing evidence from transcriptomics analyses and suggest a role for TAp73α in the regulation of cellular metabolism, cell survival, and cell growth.

Endocannabinoids stimulate human melanogenesis via type-1 cannabinoid receptor.

We show that a fully functional endocannabinoid system is present in primary human melanocytes (normal human epidermal melanocyte cells), including anandamide (AEA), 2-arachidonoylglycerol, the respective target receptors (CB(1), CB(2), and TRPV1), and their metabolic enzymes. We also show that at higher concentrations AEA induces normal human epidermal melanocyte apoptosis (∼3-fold over controls at 5 µM) through a TRPV1-mediated pathway that increases DNA fragmentation and p53 expression. However, at lower concentrations, AEA and other CB(1)-binding endocannabinoids dose-dependently stimulate melanin synthesis and enhance tyrosinase gene expression and activity (∼3- and ∼2-fold over controls at 1 µM). This CB(1)-dependent activity was fully abolished by the selective CB(1) antagonist SR141716 or by RNA interference of the receptor. CB(1) signaling engaged p38 and p42/44 mitogen-activated protein kinases, which in turn activated the cyclic AMP response element-binding protein and the microphthalmia-associated transcription factor. Silencing of tyrosinase or microphthalmia-associated transcription factor further demonstrated the involvement of these proteins in AEA-induced melanogenesis. In addition, CB(1) activation did not engage the key regulator of skin pigmentation, cyclic AMP, showing a major difference compared with the regulation of melanogenesis by α-melanocyte-stimulating hormone through melanocortin 1 receptor.

Rat and human fatty acid amide hydrolases: overt similarities and hidden differences.

Fatty acid amide hydrolase (FAAH) is a membrane protein that plays a relevant role in the metabolism of fatty acid amides and esters. It degrades important neurotransmitters such as oleamide and anandamide, and it has been involved in a number of human pathological conditions, representing therefore a valuable target for biochemical and pharmacological research. In this study, we have investigated in vitro the structure-function relationship of rat and human FAAHs. In particular circular dichroism, fluorescence spectroscopy and light scattering measurements have been performed, in order to characterize the structural features of the two proteins, both in the presence and absence of the irreversible inhibitor methoxyarachidonyl-fluorophosphonate. The results demonstrate that the structural dynamics of the two FAAHs are different, despite their high sequence homology and overall similarity in temperature-dependence. Additionally, membrane binding and kinetic assays of both FAAHs indicate that also the functional properties of the two enzymes are different in their interaction with lipid bilayers and with exogenous inhibitors. These findings suggest that pre-clinical studies of FAAH-dependent human diseases based only on animal models should be interpreted with caution, and that the efficacy of new drugs targeted to FAAH should be tested in vitro, on both rat and human enzymes.

Anandamide inhibits metabolism and physiological actions of 2-arachidonoylglycerol in the striatum.

Of the endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have received the most study. A functional interaction between these molecules has never been described. Using mouse brain slices, we found that stimulation of metabotropic glutamate 5 receptors by 3,5-dihydroxyphenylglycine (DHPG) depressed inhibitory transmission in the striatum through selective involvement of 2-AG metabolism and stimulation of presynaptic CB1 receptors. Elevation of AEA concentrations by pharmacological or genetic inhibition of AEA degradation reduced the levels, metabolism and physiological effects of 2-AG. Exogenous AEA and the stable AEA analog methanandamide inhibited basal and DHPG-stimulated 2-AG production, confirming that AEA is responsible for the downregulation of the other eCB. AEA is an endovanilloid substance, and the stimulation of transient receptor potential vanilloid 1 (TRPV1) channels mimicked the effects of endogenous AEA on 2-AG metabolism through a previously unknown glutathione-dependent pathway. Consistently, the interaction between AEA and 2-AG was lost after pharmacological and genetic inactivation of TRPV1 channels.

Anandamide extends platelets survival through CB(1)-dependent Akt signaling.

Platelets are stored at 22 degrees C, since incubation at 37 degrees C results in loss of viability. Nonetheless, in our body (37 degrees C), platelets survive for 8-10 days. This discrepancy has been explained in terms of deprivation of viability factors or accumulation of apoptotic factors during storage. We report that the endocannabinoid anandamide (AEA) may be one of the agents allowing platelet survival. In fact, at 37 degrees C, human platelets enhance the expression of pro-apoptotic proteins (caspases, Bax, Bak) and decrease the expression of Bcl-xL, thus changing the Bcl-xL/Bak ratio, a key platelet biological clock. AEA or its non-hydrolyzable analogue, methanandamide, extend platelet life span, without reversing the changes in Bcl-xL/Bak ratio induced by heat stress. Instead, AEA binding to type-1 cannabinoid receptor activates Akt, which regulates, through phosphorylation of Bad, the interactions among different Bcl-2 family members. These findings could have implications for platelet collection and, potentially, for their clinical use.

Allostery: The Rebound of Proteins.

The discovery of hemoglobin allosteric properties is briefly summarized and contextualized in the frame of the main biochemical revelations that characterized the first half of the XX century. In particular, the historical background of DNA, RNA, and protein structure research is recalled and the new role that protein-protein interaction may have on allosteric regulation is discussed.

Protein Assembly: Defining the Strength of Protein-Protein Interactions Coupling the Theory with Experiments.

In this paper we report a procedure to analyze protein homodimer interfaces.We approached the problem by means of a topological methodology. In particular, we analyzed the subunits interface of about 50 homodimers and we have defined a few parameters that allow to organize these proteins in six different classes. The main characteristics of each class of homodimers have been discussed also taking into account their stabilization energy, as reported in the literature from the experimental measurements. A paradigmatic example for each class has been reported and a graphical representation proposed in order to better explain the meaning of the parameters chosen.

Pitfalls and solutions in assaying anandamide transport in cells.

Nonspecific binding of anandamide to plastic exhibits many features that could be mistaken as biological processes, thereby representing an important source of conflicting data on the uptake and release of this lipophilic substance. Herein, we propose an improved method to assay anandamide transport, by using glass slides (i.e., coverslips) as physical support to grow cells. Although the results obtained using plastic do not differ significantly from those obtained using glass, the new procedure has the advantage of being faster, simpler, and more accurate. In fact, the lack of aspecific adsorption of anandamide to the glass surface yields a lower background and a higher precision and accuracy in determining transport kinetics, especially for the export process. Remarkably, the kinetic parameters of anandamide uptake obtained with the old and the new procedures may be similar or different depending on the cell type, thus demonstrating the complexity of the interference of plastic on the transport process. In addition, the novel procedure is particularly suitable for visualization and measurement of anandamide transport in intact cells by using a biotinylated derivative in confocal fluorescence microscopy.

Characterization of the endocannabinoid system in human neuronal cells and proteomic analysis of anandamide-induced apoptosis.

Anandamide (AEA) is an endogenous agonist of type 1 cannabinoid receptors (CB1R) that, along with metabolic enzymes of AEA and congeners, compose the "endocannabinoid system." Here we report the biochemical, morphological, and functional characterization of the endocannabinoid system in human neuroblastoma SH-SY5Y cells that are an experimental model for neuronal cell damage and death, as well as for major human neurodegenerative disorders. We also show that AEA dose-dependently induced apoptosis of SH-SY5Y cells. Through proteomic analysis, we further demonstrate that AEA-induced apoptosis was paralleled by an approximately 3 to approximately 5-fold up-regulation or down-regulation of five genes; IgG heavy chain-binding protein, stress-induced phosphoprotein-1, and triose-phosphate isomerase-1, which were up-regulated, are known to act as anti-apoptotic agents; actin-related protein 2/3 complex subunit 5 and peptidylprolyl isomerase-like protein 3 isoform PPIL3b were down-regulated, and the first is required for actin network formation whereas the second is still function-orphan. Interestingly, only the effect of AEA on BiP was reversed by the CB1R antagonist SR141716, in SH-SY5Y cells as well as in human neuroblastoma LAN-5 cells (that express a functional CB1R) but not in SK-NBE cells (which do not express CB1R). Silencing or overexpression of BiP increased or reduced, respectively, AEA-induced apoptosis of SH-SY5Y cells. In addition, the expression of BiP and of the BiP-related apoptotic markers p53 and PUMA was increased by AEA through a CB1R-dependent pathway that engages p38 and p42/44 mitogen-activated protein kinases. Consistently, this effect of AEA was minimized by SR141716. In conclusion, we identified BiP as a key protein in neuronal apoptosis induced by AEA.

Expression of the endocannabinoid system in the bi-potential HEL cell line: commitment to the megakaryoblastic lineage by 2-arachidonoylglycerol.

The role of the endocannabinoid system in haematopoietic cells is not completely understood. We investigated whether human erythroleukemia (HEL) cells were able to bind, metabolise and transport the main endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG). We also investigated whether AEA or 2-AG could modulate HEL differentiation. Although able to internalise both endocannabinoids, HEL cells had the machinery to metabolise 2-AG only, since they were devoid of the enzymes needed to synthesise and degrade AEA. Nonetheless, the intracellular transport of exogenous AEA might be required to activate the vanilloid receptors, with yet unknown implications for vascular biology. On the contrary, 2-AG appeared to play a role in lineage determination. Indeed, 2-AG itself drove HEL cells towards megakaryocytic differentiation, as it enhanced expression of beta3 integrin subunit, a megakaryocyte/platelet surface antigen, and glycoprotein VI, a late marker of megakaryocytes; in parallel, it reduced the amount of messenger RNA encoding for glycophorin A, a marker of erythroid phenotype. All these effects were mediated by activation of CB(2) cannabinoid receptors that triggered an extracellular signal-regulated kinase-dependent signalling cascade. In addition, classical inducers of megakaryocyte differentiation reduced 2-AG synthesis (although they did not affect the binding efficiency of CB(2) receptors), suggesting that levels of this endocannabinoid may be critical for committing HEL cells towards the megakaryocytic lineage.

Lipid rafts regulate 2-arachidonoylglycerol metabolism and physiological activity in the striatum.

Several G protein-associated receptors and synaptic proteins function within lipid rafts, which are subdomains of the plasma membranes that contain high concentrations of cholesterol. In this study we addressed the possible role of lipid rafts in the control of endocannabinoid system in striatal slices. Disruption of lipid rafts following cholesterol depletion with methyl-beta-cyclodestrin (MCD) failed to affect synthesis and degradation of anandamide, while it caused a marked increase in the synthesis and concentration of 2-arachidonoylglycerol (2-AG), as well as in the binding activity of cannabinoid CB1 receptors. Surprisingly, endogenous 2-AG-mediated control of GABA transmission was not potentiated by MCD treatment and, in contrast, neither basal nor 3,5-Dihydroxyphenylglycine-stimulated 2-AG altered GABA synapses in cholesterol-depleted slices. Synaptic response to the cannabinoid CB1 receptor agonist HU210 was however intact in MCD-treated slices, indicating that reduced sensitivity of cannabinoid CB1 receptors does not explain why endogenous 2-AG is ineffective in inhibiting striatal GABA transmission after cholesterol depletion. Confocal microscopy analysis suggested that disruption of raft integrity by MCD might uncouple metabotropic glutamate 5-CB1 receptor interaction by altering the correct localization of both receptors in striatal neuron elements. In conclusion, our data indicate that disruption of raft integrity causes a complex alteration of the endocannabinoid signalling in the striatum.

The endocannabinoid system in targeting inflammatory neurodegenerative diseases.

The classical divide between degenerative and inflammatory disorders of the CNS is vanishing as accumulating evidence shows that inflammatory processes are important in the pathophysiology of primarily degenerative disorders, and neurodegeneration complicates primarily inflammatory diseases of the brain and spinal cord. Here, we review the contribution of degenerative and inflammatory processes to CNS disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis and HIV-associated dementia. An early combination of neuroprotective and anti-inflammatory approaches to these disorders seems particularly desirable because isolated treatment of one pathological process might worsen another. We also discuss the apparently unique opportunity to modify neurodegeneration and neuroinflammation simultaneously by pharmacological manipulation of the endocannabinoid system in the CNS and in peripheral immune cells. Current knowledge of this system and its involvement in the above CNS disorders are also reviewed.

Properties of copper-free pig kidney amine oxidase: role of topa quinone.

Copper removal from pig kidney amine oxidase containing Cu/topaquinone (TPQ) has been obtained using CN(-) in the presence of the poor substrate p-(dimethylamino)benzylamine. Upon removal of copper, the enzyme loses its activity while the TPQ cofactor remains in its oxidized form. The addition of copper to the apo-form fully restores the active enzyme. The CN(-) treatment in the presence of sodium dithionite or good substrates (cadaverine or benzylamine) also removes copper but the TPQ cofactor is irreversibly reduced and the addition of copper does not regenerate the active enzyme. Ni(II) and Zn(II) do not bind the apo-protein in contrast to Co(II) which is incorporated to the same extent as Cu(II). However, Co-reconstituted enzyme only shows a very low activity. These results demonstrate that copper is essential for the catalytic mechanism because it maintains the correct active site geometry.

Physico-chemical properties of molten dimer ascorbate oxidase.

The possible presence of dimeric unfolding intermediates might offer a clue to understanding the relationship between tertiary and quaternary structure formation in dimers. Ascorbate oxidase is a large dimeric enzyme that displays such an intermediate along its unfolding pathway. In this study the combined effect of high pressure and denaturing agents gave new insight on this intermediate and on the mechanism of its formation. The transition from native dimer to the dimeric intermediate is characterized by the release of copper ions forming the tri-nuclear copper center located at the interface between domain 2 and 3 of each subunit. This transition, which is pH-dependent, is accompanied by a decrease in volume, probably associated to electrostriction due to the loosening of intra-subunit electrostatic interactions. The dimeric species is present even at 3 x 10(8) Pa, providing evidence that mechanically or chemically induced unfolding lead to a similar intermediate state. Instead, dissociation occurs with an extremely large and negative volume change (DeltaV approximately -200 mL.mol(-1)) by pressurization in the presence of moderate amounts of denaturant. This volume change can be ascribed to the elimination of voids at the subunit interface. Furthermore, the combination of guanidine and high pressure uncovers the presence of a marginally stable (DeltaG approximately 2 kcal.mol(-1)) monomeric species (which was not observed in previous equilibrium unfolding measurements) that might be populated in the early folding steps of ascorbate oxidase. These findings provide new aspects of the protein folding pathway, further supporting the important role of quaternary interactions in the folding strategy of large dimeric enzymes.

Regulation by cannabinoid receptors of anandamide transport across the blood-brain barrier and through other endothelial cells.

The endocannabinoid anandamide (AEA) has many neurovascular activities. However, it is not yet clear how AEA can be metabolized at the neurovascular interface, and how it can move through the vascular and the cerebral compartments. The results reported in this article show that isolated bovine brain microvessels, an ex vivo model of the blood-brain barrier, have detectable levels of endogenous AEA and possess the biochemical machinery to bind and metabolize it, i.e. type-1 and type-2 cannabinoid receptors (CB1R and CB2R), a selective AEA membrane transporter (AMT), an AEA-degrading fatty acid amide hydrolase, and the AEA-synthesizing enzymes N-acyltransferase and N-acyl-phosphatidylethanolamines-specific phospholipase D. We also show that activation of CB1R enhances AMT activity through increased nitric oxide synthase (NOS) activity and subsequent increase of NO production. AMT activity is instead reduced by activation of CB2R, which inhibits NOS and NO release. In addition, binding experiments and immunoelectronmicroscopy demonstrate that different endothelial cells vary in the expression of CB1R and CB2R on the luminal and/or abluminal sides. The different localization of CBRs can lead to a diverse effect on AMT activity on the luminal and abluminal membranes, suggesting that the distribution of these receptors may drive AEA directional transport through the blood-brain barrier and other endothelial cells.

The endocannabinoid system in neurodegeneration.

Endocannabinoids are bioactive lipids, that comprise amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the best studied endocannabinoids, and act as agonists of cannabinoid receptors. Thus, AEA and 2-AG mimic several pharmacological effects of the exogenous cannabinoid delta9-tetrahydrocannabinol, the psychoactive principle of hashish and marijuana. It is known that the activity of endocannabinoids at their receptors is limited by cellular uptake through specific membrane transporters, followed by intracellular degradation by a fatty acid amide hydrolase (for AEA and partly 2-AG) or by a monoacylglycerol lipase (for 2-AG). Together with AEA, 2-AG and congeners, the proteins that bind, transport and metabolize these lipids form the "endocannabinoid system". This new system will be briefly presented in this review, in order to put in a better perspective the role of the endocannabinoid pathway in neurodegenerative disorders, like Parkinson's disease, Huntington's disease, and multiple sclerosis. In addition, the potential exploitation of antagonists of endocannabinoid receptors, or of inhibitors of endocannabinoid metabolism, as next-generation therapeutics will be discussed.

Experimental parkinsonism alters endocannabinoid degradation: implications for striatal glutamatergic transmission.

Cannabinoid receptors and their endogenous ligands have been recently identified in the brain as potent inhibitors of neurotransmitter release. Here we show that, in a rat model of Parkinson's disease induced by unilateral nigral lesion with 6-hydroxydopamine (6-OHDA), the striatal levels of anandamide, but not that of the other endocannabinoid 2-arachidonoylglycerol, were increased. Moreover, we observed a decreased activity of the anandamide membrane transporter (AMT) and of the anandamide hydrolase [fatty acid amide hydrolase (FAAH)], whereas the binding of anandamide to cannabinoid receptors was unaffected. Spontaneous glutamatergic activity recorded from striatal spiny neurons was higher in 6-OHDA-lesioned rats. Inhibition of AMT by N-(4-hydroxyphenyl)-arachidonoylamide (AM-404) or by VDM11, or stimulation of the cannabinoid CB1 receptor by HU-210 reduced glutamatergic spontaneous activity in both naive and 6-OHDA-lesioned animals to a similar extent. Conversely, the FAAH inhibitors phenylmethylsulfonyl fluoride and methyl-arachidonoyl fluorophosphonate were much more effective in 6-OHDA-lesioned animals. The present study shows that inhibition of anandamide hydrolysis might represent a possible target to decrease the abnormal cortical glutamatergic drive in Parkinson's disease.

The importance of being dimeric.

Why are there so many dimeric proteins and enzymes? While for heterodimers a functional explanation seems quite reasonable, the case of homodimers is more puzzling. The number of homodimers found in all living organisms is rapidly increasing. A thorough inspection of the structural data from the available literature and stability (measured from denaturation-renaturation experiments) allows one to suggest that homodimers can be divided into three main types according to their mass and the presence of a (relatively) stable monomeric intermediate in the folding-unfolding pathway. Among other explanations, we propose that an essential advantage for a protein being dimeric may be the proper and rapid assembly in the cellular milieu.

Creating conditions similar to those that occur during exposure of cells to microgravity induces apoptosis in human lymphocytes by 5-lipoxygenase-mediated mitochondrial uncoupling and cytochrome c release.

Creating conditions similar to those that occur during exposure of cells to microgravity induced a sixfold increase of apoptotic bodies and DNA fragments in human lymphocytes, paralleled by an early (within 2 h) fourfold increase in 5-lipoxygenase (5-LOX) activity and a fivefold decrease in mitochondrial membrane potential and increase in cytochrome c release (within 4 and 8 h, respectively). Similar membrane potential and cytochrome c release were observed in isolated mitochondria treated with physiological amounts of 5-LOX and were enhanced by creating conditions similar to those that occur during exposure of cells to microgravity. 5-LOX inhibitors, 5,8,11,14-eicosatetraynoic acid and caffeic acid, completely prevented apoptosis, whereas the phospholipase A(2) inhibitor methyl-arachidonoyl fluorophosphonate and the 5-LOX activating protein inhibitor MK886 reduced it to 65-70%. The intracellular calcium chelator EGTA-acetoxymethylester reduced 5-LOX activity and apoptosis to 30-40% of controls, whereas the p38 mitogen-activated protein kinase inhibitor SB203580 was ineffective. The caspase-3 and caspase-9 inhibitors Z-Asp(OCH(3))-Glu(OCH(3))-Val-Asp(OCH(3))-fluoromethylketone (FMK) and Z-Leu-Glu(OCH(3))-His-Asp(OCH(3))-FMK reduced apoptotic bodies to 25-30% of the control cells. Finally, creating conditions similar to those that occur during exposure of cells to microgravity did not induce apoptosis in human lymphoma U937 cells, which did not express an active 5-LOX.