Gennaro Marino

Melusin, a muscle-specific integrin β1-interacting protein, is required to prevent cardiac failure in response to chronic pressure overload

Cardiac hypertrophy is an adaptive response to a variety of mechanical and hormonal stimuli, and represents an early event in the clinical course leading to heart failure. By gene inactivation, we demonstrate here a crucial role of melusin, a muscle-specific protein that interacts with the integrin β1 cytoplasmic domain, in the hypertrophic response to mechanical overload. Melusin-null mice showed normal cardiac structure and function in physiological conditions, but when subjected to pressure overload—a condition that induces a hypertrophic response in wild-type controls—they developed an abnormal cardiac remodeling that evolved into dilated cardiomyopathy and contractile dysfunction. In contrast, the hypertrophic response was identical in wild-type and melusin-null mice after chronic administration of angiotensin II or phenylephrine at doses that do not increase blood pressure—that is, in the absence of cardiac biomechanical stress. Analysis of intracellular signaling events induced by pressure overload indicated that phosphorylation of glycogen synthase kinase-3β (GSK-3β) was specifically blunted in melusin-null hearts. Thus, melusin prevents cardiac dilation during chronic pressure overload by specifically sensing mechanical stress.

Emilin1 links TGF-β maturation to blood pressure homeostasis

TGF-beta proteins are main regulators of blood vessel development and maintenance. Here, we report an unprecedented link between TGF-beta signaling and arterial hypertension based on the analysis of mice mutant for Emilin1, a cysteine-rich secreted glycoprotein expressed in the vascular tree. Emilin1 knockout animals display increased blood pressure, increased peripheral vascular resistance, and reduced vessel size. Mechanistically, we found that Emilin1 inhibits TGF-beta signaling by binding specifically to the proTGF-beta precursor and preventing its maturation by furin convertases in the extracellular space. In support of these findings, genetic inactivation of Emilin1 causes increased TGF-beta signaling in the vascular wall. Strikingly, high blood pressure observed in Emilin1 mutants is rescued to normal levels upon inactivation of a single TGF-beta1 allele. This study highlights the importance of modulation of TGF-beta availability in the pathogenesis of hypertension.

Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma 'anandamide amidohydrolase'.

The endogenous cannabimimetic substance, anandamide (N‐arachidonoyl‐ethanolamine) and the recently isolated sleep‐inducing factor, oleoyl‐amide (cis‐9,10‐octadecenoamide), belong to two neuroactive fatty acid amide classes whose action in mammals has been shown to be controlled by enzymatic amide bond hydrolysis. Here we report the partial characterisation and purification of ‘anandamide amidohydrolase’ from membrane fractions of N18 neuroblastoma cells, and provide evidence for a further and previously unsuspected role of this enzyme. An enzymatic activity catalysing the hydrolysis of [14C]anandamide was found in both microsomal and 10,000 × g pellet fractions. The latter fractions, which displayed the highest V max for anandamide, were used for further characterisation of the enzyme, and were found to catalyse the hydrolysis also of [14C]oleoyl‐amide, with an apparent K m of 9.0 ± 2.2 μM. [14C]anandamide‐ and [14C]oleoyl‐amide‐hydrolysing activities: (i) exhibited identical pH‐ and temperature‐dependency profiles; (ii) were inhibited by alkylating agents; (iii) were competitively inhibited by the phospholipase A2 inhibitor arachidonyl‐trifluoromethyl‐ketone with the same IC50 (3 μM); (iv) were competitively inhibited by both anandamide (or other polyunsaturated fatty acid‐ethanolamides) and oleoyl‐amide. Proteins solubilised from 10,000 × g pellets were directly analysed by isoelectric focusing, yielding purified fractions capable of catalysing the hydrolysis of both [14C]anandamide and [14C]oleoyl‐amide. These data suggest that ‘anandamide amidohydrolase’ enzymes, such as that characterised in this study, may be used by neuronal cells also to hydrolyse the novel sleep‐inducing factor oleoyl‐amide.

Syringopeptins, new phytotoxic lipodepsipeptides of Pseudomonas syringae pv. syringae

The primary structure of some new lipodepsipeptides named syringopeptins, produced by plant pathogenic strains of Pseudopmonas syringae pv. syringae has been determined by a combination of chemical methods, 1H and 13C NMR spectroscopy and FAB mass spectrometry. Two syringomycin‐producing strains afforded 3‐hydroxydecanoyl‐Dhb‐Pro‐Val‐Val‐Ala‐Ala‐Val‐Val‐Dhb‐Ala‐Val‐Ala‐Ala‐Dhb‐aThr‐Ser‐Ala‐Dhb‐Ala‐Dab‐Dab‐Tyr, with Tyr acylating a Thr to form a macrolactone ring, and smaller amounts of the 3‐hydroxydodecanoyl homologue. Evidence was obtained that a third syringomycin‐producing strain and a syringotoxin‐producing strain synthesize 3‐hydroxydecanoyl‐Dhb‐Pro‐Val‐Ala‐Ala‐Val‐Leu‐Ala‐Ala‐Dhb‐Val‐Dhb‐Ala‐Val‐Ala‐Ala‐Dhb‐aThr‐Ser‐Ala‐Val‐Ala‐Dab‐Dab‐Tyr, with Tyr and aThr forming again the macrolactone ring, and smaller amounts of the 3‐hydroxydodecanoyl homologue.

The structure of syringomycins A1, E and G

By a combination of 1D and 2D 1H‐ and 13C‐NMR, FAB‐MS, and chemical and enzymatic reactions carried out at the milligram level, it has been demonstrated that syringomycin E, the major phytotoxic antibiotic produced by Pseudomonas syringae pv. syringae, is a new lipodepsipeptide. Its amino acid sequence is Ser‐Ser‐Dab‐Dab‐Arg‐Phe‐Dhb‐4(Cl)Thr‐3(OH)Asp with the β‐carboxy group of the C‐terminal residue closing a macrocyclic ring on the OH group of the N‐terminal Ser, which in turn is N‐acylated by 3‐hydroxydodecanoic acid. Syringomycins A1 and G, two other metabolites of the same bacterium, differ from syringomycin E only in their fatty acid moieties corresponding, respectively, to 3‐hydroxydecanoic and 3‐hydroxytetradecanoic acid.

Protection from angiotensin II–mediated vasculotoxic and hypertensive response in mice lacking PI3Kγ

Hypertension affects nearly 20% of the population in Western countries and strongly increases the risk for cardiovascular diseases. In the pathogenesis of hypertension, the vasoactive peptide of the renin-angiotensin system, angiotensin II and its G protein–coupled receptors (GPCRs), play a crucial role by eliciting reactive oxygen species (ROS) and mediating vessel contractility. Here we show that mice lacking the GPCR-activated phosphoinositide 3-kinase (PI3K)γ are protected from hypertension that is induced by administration of angiotensin II in vivo. PI3Kγ was found to play a role in angiotensin II–evoked smooth muscle contraction in two crucial, distinct signaling pathways. In response to angiotensin II, PI3Kγ was required for the activation of Rac and the subsequent triggering of ROS production. Conversely, PI3Kγ was necessary to activate protein kinase B/Akt, which, in turn, enhanced L-type Ca2+ channel–mediated extracellular Ca2+ entry. These data indicate that PI3Kγ is a key transducer of the intracellular signals that are evoked by angiotensin II and suggest that blocking PI3Kγ function might be exploited to improve therapeutic intervention on hypertension.

Purification, Characterization, and Functional Role of a Novel Extracellular Protease from Pleurotus ostreatus

ABSTRACT A new extracellular protease (PoSl; Pleurotus ostreatus subtilisin-like protease) from P. ostreatus culture broth has been purified and characterized. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, a pI of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl activity is positively affected by calcium. A 10-fold decrease in the Km value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca2+binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of P. ostreatuslaccase activity by degrading and/or activating different isoenzymes.

Xylanase from the psychrophilic yeast Cryptococcus adeliae.

Abstract A xylanase belonging to family 10 is produced by Cryptococcus adeliae, an Antarctic yeast that exhibits optimal growth at low temperature. The mature glycosylated xylanase secreted by C. adeliae is composed of 338 amino acid residues and 26 ± 3 osidic residues, and shares 84% identity with its mesophilic counterpart from C. albidus. The xylanase from C. adeliae is less thermostable than its mesophilic homologue when the residual activities are compared, and this difference was confirmed by differential scanning calorimetry experiments. In the range 0°–20°C, the cold-adapted xylanase displays a lower activation energy and a higher catalytic efficiency. All these observations suggest a less compact, more flexible molecular structure. Analysis of computerized molecular models built up for both psychrophilic and mesophilic xylanases indicates that the adaptation to cold consists of discrete changes in the tridimensional structure: of 53 substitutions, 22 are presumably involved in the adaptation process. These changes lead mainly to a less compact hydrophobic packing, to the loss of one salt bridge, and to a destabilization of the macrodipoles of the helices.

Cooperation Between Insulin and Leptin in the Modulation of Vascular Tone

Abstract—High levels of insulin and leptin have been reported in human hypertension, suggesting a role for these metabolic hormones in blood pressure homeostasis. These hormones interact on intermediate metabolism, but nothing is known about their interaction at the vascular level. Our data demonstrate that insulin (0.6 nmol/L) is able to enhance vasodilation induced by leptin (10−11 to 10−6 mol/L; percentage change in maximal vasodilation, 39±3% vs 26±2%; n=6, P <0.03) but not by acetylcholine. Moreover, we demonstrate by 4,5-diaminofluorescein (DAF)-2 that insulin potentiates leptin-induced nitric oxide (NO) release. Finally, Western blotting studies show that insulin enhances the leptin-induced phosphorylation of Akt in Ser473 and Thr308 and of endothelial NO synthase in Ser1177. In conclusion, our data demonstrate that insulin and leptin cooperate in the modulation of vascular tone through enhancement of endothelial NO release. This phenomenon could have a major impact on the regulation of the cardiovascular system, principally in those clinical conditions characterized by endothelial NO dysfunction and metabolic disorders, such as arterial hypertension.

Characterisation of S-nitrosohaemoglobin by mass spectrometry

Recent studies have demonstrated the biological importance of the interaction of S‐nitrosothiols, which can be considered as nitric oxide (NO) protein donors, especially haemoglobin, at the level of Cys residues. It was recently proposed that S‐nitrosohaemoglobin is formed within red blood cells and serves as a regulatory function. In human haemoglobin the α‐subunit contains one Cys residue and the β‐subunit contains two Cys residues, one of which (β‐Cys93) is highly reactive and conserved among species, although its function has remained unknown. Electrospray ionization mass spectrometry was used to monitor the results of exposure of haemolysates to S‐nitrosocysteine under different conditions and thus addressed some aspects of NO–haemoglobin interaction. When an equimolar ratio of S‐nitrosothiol was added to haemoglobin, only a single NO molecule was added. Peptide mapping by liquid chromatography–mass spectrometry located the nitrosyl group at the level of β‐Cys93 demonstrating that this was the preferred site of formation of S‐nitrosohaemoglobin. The present data also suggest that electrospray mass spectrometry can allow quantification and characterisation of S‐nitrosoproteins in blood.