Three-dimensional structure of acylphosphatase. Refinement and structure analysis.

We report here the complete determination of the solution structure of acylphosphatase, a small enzyme that catalyses the hydrolysis of organic acylphosphates, as determined by distance geometry methods based on nuclear magnetic resonance information. A non-standard strategy for the distance geometry calculations was used and is described here some detail. The five best structures were then refined by restrained energy minimization and molecular dynamics in order to explore the conformational space consistent with the experimental data. We address the question of whether the solution structure of acylphosphatase follows the general principles of protein structure, i.e. those learned from analysing crystal structures. Static and dynamic features are discussed in detail. An uncommon beta-alpha-beta motif, so far found only in procarboxypeptidase B and in an RNA-binding protein, is present in acylphosphatase.     

The regions of the sequence most exposed to the solvent within the amyloidogenic state of a protein initiate the aggregation process

Formation of misfolded aggregates is an essential part of what proteins can do. The process of protein aggregation is central to many human diseases and any aggregating event needs to be prevented within a cell and in protein design. In order to aggregate, a protein needs to unfold its native state, at least partially. The conformational state that is prone to aggregate is difficult to study, due to its aggregating potential and heterogeneous nature. Here, we use a systematic approach of limited proteolysis, in combination with electrospray ionisation mass spectrometry, to investigate the regions that are most flexible and solvent-exposed within the native, ligand-bound and amyloidogenic states of muscle acylphosphatase (AcP), a protein previously shown to form amyloid fibrils in the presence of trifluoroethanol. Seven proteases with different degrees of specificity have been used for this purpose. Following exposure to the aggregating conditions, a number of sites along the sequence of AcP become susceptible to proteolytic digestion. The pattern of proteolytic cleavages obtained under these conditions is considerably different from that of the native and ligand-bound conformations and includes a portion within the N-terminal tail of the protein (residues 6-7), the region of the sequence 18-23 and the position 94 near the C terminus. There is a significant overlap between the regions of the sequence found to be solvent-exposed from the present study and those previously identified to be critical in the rate-determining steps of aggregation from protein engineering approaches. This indicates that a considerable degree of solvent exposure is a feature of the portions of a protein that initiate the process of aggregation.     

During muscle ageing the activation of the mitogenic signalling is not sufficient to guarantee cellular duplication

Satellite cells are quiescent cells that can be induced to proliferate by a variety of stimuli such as injury and exercise, providing in this way a source of new myoblasts that repopulate the damaged muscle. It is well known that, as senescence progresses, the muscle regenerative potential progressively diminishes, but the molecular mechanisms underlying this process are not yet completely defined. Many growth factors, including Platelet Derived Growth Factor (PDGF-BB)*, have been associated to satellite cells activation, acting as potent mitogenic agents for these cells. The aim of this study is to explore if the diminished response of senescent myoblasts to growth stimuli could be due to the inability to receive and transduce hormonal signals. Herein, we demonstrate that that although PDGF-r expression is down-regulated during senescence, the receptor is fully able to be phosphorylated and to transmit the signal. Although senescent myoblasts display increased level of phosphotyrosine phosphatases (PTPs), neither the PDGF receptor (PDGF-r) phosphorylation level nor the citosolic signal transduction machinery is affected. Indeed, we demonstrated that senescent human myoblasts are able to initiate a proper mitogenic signalling cascade, since the activation of mitogen-activated protein kinases (MAPK) and phosphatydil inositole 3 kinase (PI-3K) pathways is similar in young and senescent cells. Our data underline that, despite a conserved capability to activate PDGF-r after agonist stimulation and a functional signal transduction machinery, the mitogenic signal initiated by growth factors in senescent cells does not lead to cell division, being unable to overcome the cell cycle block, likely caused by the accumulation of the inhibitor p21WAF1.     

Up-regulated expression of low molecular weight protein tyrosine phosphatases in different human cancers

Protein tyrosine phosphorylation, mediated by the balanced action of tyrosine kinases and phosphatases, contributes to the regulation of the growth, migration, and invasion of normal and malignant cells. Among tyrosine phosphatases, low molecular weight protein tyrosine phosphatases (LMW-PTP) have been recognized as a possible "positive factor" in tumour onset and progression. The aim of this work was to assess whether LMW-PTP are differentially expressed in normal and malignant tissues. Using real-time PCR analysis we evaluated the expression levels of total LMW-PTP mRNA in surgical samples of breast, colon and lung cancers (63, 60, and 58, respectively), and in their paired adjacent not affected tissues. Moreover, the same analysis was carried out on a group of neuroblastomas (25 cases). Significant correlations between LMW-PTP overexpression and the most common clinical-pathological features of cancers exist. In colon cancer and neuroblastoma increased total LMW-PTP mRNA expression correlates with unfavourable outcome. While LMW-PTP mRNA expression increases in tumour samples, the relative contribution of the different isoforms does not change. Our findings indicate that LMW-PTP can be considered an oncogene as it is overexpressed in different tumour types and suggests that LMW-PTP enhanced expression is generally prognostic for a more aggressive cancer.     

SNARE complexes and neuroexocytosis: how many, how close?

Regulated secretion is an essential process in all eukaryotic cells. The release of molecules contained inside exocytic granules and synaptic vesicles is mediated by the assembly of a SNARE complex formed by the coil-coiling of three proteins: SNAP-25, syntaxin and VAMP/synaptobrevin. It seems that SNARE complexes assemble together in rosette-shaped super-complexes but there is controversy on the actual number (N) of copies of SNARE complexes that are necessary to mediate exocytosis. We discuss attempts to determine the value of N and suggest that N varies with the type of exocytic vesicles. In addition, we propose that the N value in neuroexocytosis can be estimated by the comparative use of different types of botulinum neurotoxins.     

Insulin inhibits platelet-derived growth factor-induced cell proliferation

Cellular behavior can be considered to be the result of a very complex spatial and temporal integration of intracellular and extracellular signals. These signals arise from serum-soluble factors as well as from cell-substrate or cell-cell interactions. The current approach in mitogenesis studies is generally to analyze the effect of a single growth factor on serum-starved cells. In this context, a metabolic hormone such as insulin is found to be a mitogenic agent in many cellular types. In the present study, we have considered the effect of insulin stimulation in platelet-derived growth factor (PDGF)-activated NIH-3T3 and C2C12 cells. Our results show that insulin is able to inhibit strongly both NIH-3T3 and C2C12 cell growth induced by PDGF, one of the most powerful mitotic agents for these cell types. This inhibitory effect of insulin is due primarily to a premature down-regulation of the PDGF receptor. Thus, when NIH-3T3 or C2C12 cells are stimulated with both PDGF and insulin, we observe a decrease in PDGF receptor phosphorylation with respect to cells treated with PDGF alone. In particular, we find that costimulation with insulin leads to a reduced production of H2O2 with respect to cell stimulation with PDGF alone. The relative low concentration of H2O2 in PDGF/insulin-costimulated cell leads to a limited down-regulation of protein tyrosine phosphatases, and, consequently, to a reduced PDGF receptor phosphorylation efficiency. The latter is very likely to be responsible for the insulin-dependent inhibition of PDGF-receptor mitogenic signaling.     

Phosphatidylinositol 3-kinase C2alpha is essential for ATP-dependent priming of neurosecretory granule exocytosis

Neurotransmitter release and hormonal secretion are highly regulated processes culminating in the calcium-dependent fusion of secretory vesicles with the plasma membrane. Here, we have identified a role for phosphatidylinositol 3-kinase C2alpha (PI3K-C2alpha) and its main catalytic product, PtdIns3P, in regulated exocytosis. In neuroendocrine cells, PI3K-C2alpha is present on a subpopulation of mature secretory granules. Impairment of PI3K-C2alpha function specifically inhibits the ATP-dependent priming phase of exocytosis. Overexpression of wild-type PI3K-C2alpha enhanced secretion, whereas transfection of PC12 cells with a catalytically inactive PI3K-C2alpha mutant or a 2xFYVE domain sequestering PtdIns3P abolished secretion. Based on these results, we propose that production of PtdIns3P by PI3K-C2alpha is required for acquisition of fusion competence in neurosecretion.     

Integrated Assessment of Large-Scale Biofuel Production

This paper proposes a different framework for discussing the possibility of replacing a significant fraction of fossil energy consumption of modern economies with biofuels. The proposed analysis is not based on the two classic feasibility checks—land availability and output/input energy ratio—debated in the majority of the literature in this field. Rather, the focus is on the desirability of an energy sector powered by biomass energy. Discussing of desirability requires introducing a multicriteria approach, that in turn requires a definition of a set of criteria of performance for such an energy sector. The concepts of societal metabolism and ecosystem metabolism are introduced to define five criteria of performance for an energy sector powered by alternative sources.

This paper does not tell the society whether or not biofuels should be used to replace fossil energy. Rather, it proposes a method of characterization of pros and cons for the option biofuel which can be used to make more informed choices. An analysis of three systems of production—corn-ethanol, sunflower-biodiesel, and SRWC-methanol—is provided to indicate the existence of systemic characteristics associated with an energy sector powered by biofuels. These characteristics are likely to persist even when different technical coefficients will be achieved. The conclusion is that, at the moment, it is not possible to replace the actual performance of an energy sector based on fossil energy with an energy sector running on biofuel. Biomass energy can and will have to play an important role in the sustainability of humankind, but this will require a better understanding of (i) the role that an energy sector plays within a given structure of societal metabolism; and (ii) the impact generated on ecosystem metabolism by societal metabolism, plus a lot of wisdom.     

Erythrocyte sodium/hydrogen exchange inhibition by (-) epicatechin

Epicatechin, a flavonoid belonging to the group of compounds collectively called catechins, have been reported to possess insulin-like properties. Besides their anti-diabetic properties, catechins also show growth inhibition. Since cytosolic pH (pHi) plays a role in cell proliferation and the Na/H exchanger (NHE) is the major pH (pHi) regulatory mechanism, we undertook in vitro studies with human erythrocytes to examine the effect of (-) epicatechin (EC) on the NHE1 isoform. NHE activity was measured in eight healthy volunteers, eight type 1 diabetics, and nine type 2 diabetics, following 30 min incubations at 37 degrees C with either 1 mM epicatechin, 10(-9) M insulin or solvent alone. NHE activity was elevated in both groups of patients (P< 0.05). Epicatechin caused a 93% decrease in Na/H antiport activity in health controls, 89 and 86% in type 1 and type 2 diabetics, respectively (P< 0.001). Insulin caused a 36% decrease in antiport activity only in the type 2 diabetic group (P< 0.05). The strong inhibition of erythrocyte NHE1 (the ubiquitously present isoform) by epicatechin may have important implications. NHE1 inhibition could be one of the major mechanisms underlying the antiproliferative effects of catechins.